Replication factor C-40 (RFC40/RFC2) as a prognostic marker and target in estrogen positive and negative and triple negative breast cancer

ABSTRACT

The present disclosure relates generally to cancer and particularly to breast cancer including estrogen sensitive, estrogen resistant and triple negative breast cancer (TNBC), and to methods of diagnosis and prognosis thereof and therapeutic intervention involving replication factor C 40 (RFC40). Methods and assays for evaluating breast cancer are provided. The disclosure also relates to inhibition or modulation of RFC40 in treatment or alleviation of cancer, including breast cancer. RFC40 inhibitors, including siRNAs, miRNAs, and shRNAs, which specifically affect cancer cells, particularly breast cancer cells, are provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/948,896, filed on Nov. 23, 2015, now allowed, which is acontinuation-in-part of U.S. application Ser. No. 14/204,639, filed Mar.11, 2014, now U.S. Pat. No. 9,193,970, issued on Nov. 24, 2015, whichclaims priority to U.S. application No. 61/782,901, filed Mar. 14, 2013,the disclosures of each of which are incorporated herein by reference.

FIELD

The present invention relates generally to cancer and particularly tobreast cancer including estrogen sensitive, estrogen resistant andtriple negative breast cancer (TNBC), and to methods of diagnosis andprognosis thereof and therapeutic intervention involving replicationfactor C 40 (RFC40). The invention also relates to inhibition ormodulation of RFC40 in treatment or alleviation of cancer, includingbreast cancer.

BACKGROUND OF THE INVENTION

Breast cancer accounts for 18% of all cancers in women, making it theforemost cause of cancer-related deaths in women (McPherson K et al(2000) BMJ 321(7261):624-8). Currently, routine mammography is the mostcommonly used method for early detection of breast cancer (Smith R A etal (2012) Oncology 26(5):471-5, 479-81, 485-6). Therefore, earlydiagnosis and treatment of breast cancer could play a monumental role inreducing deaths (Misek D E and Kim E H (2011) Int J Proteomics2011:343582). Most of the drugs available for the treatment of breastcancers target growth factor and endocrine receptors, particularly theendocrine (estrogen; ER) or growth factor ((ErbB-1, ErbB-2 [humanepidermal growth factor receptor 2; HER2], ErbB-3 and ErbB-4) receptorsfor therapy (Normanno N et al (2009) Endocr Relat Cancer 16(3):675-702).

However, emerging resistance to endocrine drugs and therapies targetedagainst HER2 receptors have created a dire need for identification ofmolecular targets that are non-receptor based and directly involved inthe proliferation of the cancer cells (Normanno N et al (2005) EndocrRelat Cancer 12(4):721-47; Normanno N et al (2009) Endocr Relat Cancer16(3):675-702). Triple Negative breast cancer (TNBC) is known to be themost aggressive of breast cancers that can metastasis beyond the breastand are more likely to recur after treatment. Tumors and cells of thissubtype of breast cancer lack the estrogen, progesterone as well as thehuman epidermal growth factor receptor 2 and hence will not respond tothe traditional therapies. Although, estrogen positive and HER2over-expressed breast cancers have relatively good target-based agentsfor treatment, Triple Negative Breast cancer (TNBC) will not respond tothese therapies since it lacks all these receptors. There is therefore ahuge void for therapies for patients with triple-negative breast cancer(endocrine and growth receptor negative). Hence, the discovery of nonreceptor based target therapies that may be universally applicable toall sub-types of breast cancers is of paramount importance.

DNA replication is one of the most remarkable and challenging steps inthe cell cycle and requires the collaboration of a formidable number ofproteins. In eukaryotes, several accessory proteins such as ReplicationFactor C (RFC) and Proliferating Cell Nuclear Antigen (PCNA), conferspeed and high processivity to the replicative polymerases, DNApolymerases δ (Pol δ) and ε. The RFC loads PCNA onto DNA and consists offive subunits, RFC140, RFC40, RFC38, RFC37 and RFC36 (Gupte R S et al(2005) Cell Cycle 4(2): 323-329). Its assembly commits the cell to DNAreplication and is involved in many DNA transactions such as DNA damagecheckpoint response, maintenance of genomic stability and regulation ofsister chromatid cohesion in mitosis as well as in meiosis (Majka J andBurgers P M (2004) Prog Nucleic Acid Res Mol Bio 178: 227-260;Petronczki M et al (2004) J Cell Sci 117(Pt 16): 3547-3559).

Amongst all the RFC subunits, only the second subunit, RFC40/RFC2 canindependently unload PCNA and inhibit DNA Pol δ activity (Cai J et al(1997) J Biol Chem 272(30):18974-81; Pan Z Q et al (1993) Proc Natl AcadSci USA 90(1):6-10). It has been recently discovered that RFC40 isrequired for accurate chromosomal segregation and completion of celldivision after mitosis in proliferating neonatal rat cardiac myocytes,suggesting a role for RFC40 in mitosis and cytokinesis (Ata H et al(2012) PLoS One 7(6):e39009). Additionally, it was also observed thatinhibition of endogenous RFC40 in proliferating neonatal rat cardiacmyocytes causes cell death (Ata H et al (2012) PLoS One 7(6):e39009).Consistently, it has been demonstrated that deletion of RFC40 gene isembryonically lethal in yeast (Cullmann G et al (1995) Mol Cell Bio115(9):4661-71). Also, halo-insufficiency of RFC40 causes growthretardation in Williams-Beurner syndrome (Peoples R et al (1996) Am JHum Genet 58:1370-3). Taken together these findings suggest that RFC40is essential for cell proliferation.

Interaction between the second subunit of the Replication Factor C,RFC40, and the regulatory subunit of Protein Kinase A, R1α, has beenidentified using yeast two-hybrid screening (Gupte R S et al (2005) CellCycle 4(2): 323-329). This complex has been shown to be essential forcell survival and that R1α functions as a nuclear transport protein forRFC40 via its non-conventional nuclear localization sequence (NLS)(Gupte R et al (2005) Cancer Biology and Therapy 4(4):429-437).Moreover, deletions in the RFC40 binding region on R1α, or eitherdeletion or mutations of the non-conventional NLS of R1α, leads to G1arrest, suggesting that the R1α-RFC40 complex is transported to thenucleus at the G1/S transition. Additionally, elevated intracellularcAMP levels exert transcriptional/post-transcriptional effects on mRNAlevels and a translation effect on the protein expressions of both RFC40and R1α, thereby increasing the amount of the R1α-RFC40 complexformation and hence promoting the nuclear transport of RFC40 by R1α(Gupte R et al (2006) Exper Cell Res 312:796-806).

Currently there clearly is a need for molecular targets that arenon-receptor based for use and application towards the development ofdrug therapies for breast cancers lacking endocrine and growth receptorssuch as triple negative breast cancers (TNBC) and for therapies whichare endocrine and growth factor receptor independent and thereforeapplicable to all or most forms of breast cancer. The present inventionprovides a novel independent marker and target for cancer diagnosis andtherapy, particularly including breast cancer.

The citation of references herein shall not be construed as an admissionthat such is prior art to the present invention.

SUMMARY OF THE INVENTION

In one aspect, the present invention extends to the diagnosis and/ortreatment of breast cancer in mammals, particularly in humans, usingreplication factor C 40 (RFC40) and particularly to RFC40 protein and/orgene expression as a marker in breast cancer and to RFC40 as a novel andspecific oncologic target for intervention in cancer, particularly inbreast cancer, including estrogen sensitive, estrogen resistant andtriple negative breast cancer (TNBC).

The invention extends to applications in and diagnosis, prognosis,and/or treatment of breast hyperplasia, pre-neoplastic lesions and/orductal carcinoma I situ (DCIS).

In particular, the DNA replication protein, RFC40, is presentlyidentified and validated as a non-receptor based molecular marker andspecific target for breast cancer, irrespective of its receptor status,including TNBC. The studies provided herein establish that RFC40 proteinand messenger RNA encoding it, as well as RFC40 gene copy numbers, areincreased in breast cancers, including in estrogen sensitive, estrogenresistant and TNBC. The present studies demonstrate that inhibition ormodulation of RFC40 such that its activity or expression is reduced orblocked results in reduction in cell numbers and inhibition of cellproliferation or division in estrogen positive (ER positive), estrogennegative (ER negative), and progesterone, estrogen and human epidermalgrowth factor receptor 2-HER2 negative or TNBC cells. Thus, RFC40provides a non-receptor diagnostic and prognostic marker and atherapeutic or interventional target involved in cell division andproliferation which, without intending to be constrained by theory, isindependent of growth factor and endocrine receptor status. Inhibitionor modulation of RFC40 is therefore applicable in early stage breastcancer, late stage breast cancer, on drug failure or resistance toreceptor-based therapies, and in instances of recurrence.

It will be apparent from the foregoing and the description, examples andfigures herein, that the present disclosure includes methods fordiagnosing, or aiding in a diagnosis of an individual with cancer, themethod comprising testing a biological sample from the individual anddetermining RFC40 expression and/or RFC40 gene copy number, whereingreater RFC40 expression relative to a reference, and/or greater copynumber of the RFC40 gene relative to a reference, is a diagnosis or aidsin a diagnosis that the individual has breast cancer. The disclosureincludes detecting RFC40 and/or detecting an RFC40-encoding nucleicacid, including DNA and RNA. In some embodiments, the detection isperformed by detecting a signal from a detectable label in a complex ofthe protein and a detectably labeled ligand, or by detecting a signalfrom a detectable label in a complex of an RFC40 encoding polynucleotideand a detectably labeled probe.

In embodiments, RFC40-positive cancer cells, such as those that may bepresent in a tumor or other biological sample, express more RFC40protein or mRNA than a reference, and/or have an increased copy numberof the RFC40 gene than a reference. The reference can be any suitablereference, such as a matched control, a standardized value (i.e., areaunder a curve), and/or values for RFC40 mRNA or protein amountsexpressed by a cell of the same tissue type wherein the cells are notcancer cells. RFC40 gene copy number in non-malignant cells can also beused as a control. In certain embodiments identification of a humansubject as a candidate for treatment with a composition disclosed hereinis achieved using the same or similar criteria as for identifying anindividual as a candidate for therapy with other anti-cancer agents,which is based on clinically established parameters and will be known tothe skilled artisan. In certain embodiments, the individual is suspectedof having or is at risk for developing breast cancer. In certainexamples, upon diagnosing an individual as having cancer, the disclosurefurther comprises administering to the individual a pharmaceuticalcomposition of this disclosure. In another aspect, the disclosureincludes a method for treating an individual for cancer comprisingselecting an individual for treatment based on increased ReplicationFactor C-40 (RFC40) mRNA or protein expression and/or increased RFC40gene copy number relative to a control, and administering to theindividual a pharmaceutical composition of this disclosure.

In accordance with the present invention, methods for the treatment ofbreast cancer and/or the reduction of risk for breast cancer bymodulating RFC40, particularly via inhibitors specifically directedagainst RFC40, including siRNAs or miRNAs, are provided. In an aspect ofthe method, the treatment of breast cancer is provided comprisingmodulating RFC40 expression or activity in cells, particularly andspecifically in oncogenic or cancer cells versus normal or benign cells,particularly non-cancerous breast cells.

The invention thus provides a method of inhibiting the growth and/orcell division of cancer cells, said method comprising contacting apopulation of mammalian cells comprising cancer cells with an inhibitorof the activity or expression of RFC40.

In an aspect of the method, the cancer cells are breast cancer cells. Ina further aspect of the method, the breast cancer cells are estrogenpositive, estrogen negative or progesterone/estrogen/HER2 negativebreast cancer cells (TNBCs).

The method includes inhibitors of RFC40 expression and/or activity, andcompositions comprising such inhibitors, wherein the inhibitor of theactivity or expression of RFC40 is selected from the group consisting ofa small interfering RNA (siRNA), microRNA (miRNA), an antisensepolynucleotide, a ribozyme and a short-hairpin RNA (shRNA). Inembodiments the inhibitor comprises or consists of a nucleic acidsequence complementary to, or engineered from, a naturally-occurringpolynucleotide sequence. In embodiments, the nucleic acids can beapproximately 17-30 contiguous nucleotides of a nucleic acid encodingRFC40 polypeptide, or the reverse complement of a nucleic acid encodingRFC40.

The method includes in certain embodiments use of an inhibitor, whereinthe inhibitor inhibits the activity and/or expression of RFC40 andcomprises a nucleic acid sequence complementary to, or engineered from,a naturally-occurring polynucleotide sequence of about 17 to about 30contiguous nucleotides of the RFC40 nucleic acid of FIG. 18 (SEQ IDNO:2).

In a particular aspect the inhibitor is selected from a cAMP modulator,an inhibitor of CDK/cyclin E complexes, a protein kinase A (PKA)inhibitor, and an antibody against RFC40. In embodiments, the inhibitoris an indole-3 carbinole compound, olomoucine or roscovitine or8-Cl-cAMP.

Since RFC40 has been shown to interact with RIα of protein kinase A, andthis interaction facilitates or is required for nuclear translocationand activity thereby of RFC40, the inhibitor may be a compound or agentthat blocks nuclear translocation of RFC40 and/or interaction of RFC40with RIα of protein kinase A.

The invention includes a pharmaceutical composition(s) comprising amodulator, particularly an inhibitor of RFC40 for use in prophylaxisand/or therapy of cancer, particularly breast cancer. Suchpharmaceutical composition(s) may comprise an agent selected from thegroup consisting of a small interfering RNA (siRNA), microRNA (miRNA),an antisense polynucleotide, a ribozyme and a short-hairpin RNA (shRNA),wherein said agent comprises a nucleic acid sequence complementary to,or engineered from, a naturally-occurring polynucleotide sequence ofabout 17 to about 30 contiguous nucleotides of a nucleic acid sequenceencoding RFC40, and combinations thereof, and may further comprise apharmaceutically acceptable carrier, vehicle or diluent.

In embodiments, the invention includes a pharmaceutical composition(s)comprising an agent which inhibits the expression, nucleartranslocation, RIα interaction, or activity of RFC40, and apharmaceutically acceptable carrier for use in the treatment of breastcancer.

In an embodiment, the disclosure includes a pharmaceutical compositionfor use in prophylaxis and/or therapy of cancer, the compositioncomprising a pharmaceutically acceptable carrier and a polynucleotide,wherein the polynucleotide comprises a sequence selected from SEQ IDNO:3 (CUUGUAAUGCUUCGGAUAA—RFC40-siRNA-S1); SEQ ID NO:4(GAACUGCCGUGGGUUGAAA—RFC40-siRNA-S2); SEQ ID NO:5(CGGCAAGACCACAAGCAUU—RFC40-siRNA-S3); SEQ ID NO:6(GCUGUGCAGUCCUCCGGUA—RFC40-siRNA-S4); SEQ ID NO:7(ACAGGUGAGGUUCUUGGGAGCC—miR-hsa-125a-3p); SEQ ID NO:8aCAGGUGAUCCACUUGggagcc—modified miR#1); SEQ ID NO:9(aCAGGUGAGGAUAACAggagcc—modified miR#2), SEQ ID NO:18(aCAGGUGAGGUUCCUGggagcc—modified miR#3), SEQ ID NO:19(aCAGGUGAGGCUCCUGggagcc—modified miR#4), and combinations thereof. Inembodiments an shRNA can be used. In an embodiment the shRNA comprisesor consists of the sequence ACUACGAACUGCCGUGGGUUGAAAAAUAU (SEQ ID NO:15) or GUCCCGCUGUGCAGUCCUCCGGUACACAA (SEQ ID NO:16). In embodiments, thedisclosure includes an shRNA comprising the sequenceACUACGAACUGCCGUGGGUUG (SEQ ID NO: 20). In embodiments, the disclosureincludes shRNAs comprising or consisting of the following sequences:ACUACGAACUGCCGUGGGUUGNNNNNNNNNCAACCCACGGCAGUUCGUAGU (SEQ ID NO: 21),wherein N is any nucleotide, and wherein NNNNNNNNN the may form the loopof the shRNA. In an embodiment, SEQ ID NO: 21 comprises the sequenceUUCAAGAGA (SEQ ID NO:22) as the loop sequence. Thus, the disclosureincludes an shRNA comprising or consisting of the sequenceACUACGAACUGCCGUGGGUUGUUCAAGAGACAACCCACGGCAGUUCGUAGU (SEQ ID NO:23). Thisis also referred to herein as the hRFC2-shRNA#1 sequence. SEQ ID NO: 23may also include a transcription a termination sequence, such as UUUUUU,encoded by TTTTTT.

In another example, the disclosure includes an shRNA comprising orconsisting of the sequence:GUCCCGCUGUGCAGUCCUCCGGUACACAANNNNNNNNNUGUGUACCGGAGGA CUGCACAGCGGGAC (SEQID NO: 24), wherein the NNNNNNNNN may form the loop of the shRNA. In anembodiment, the NNNNNNNNN of SEQ ID NO: 24 comprises UUCAAGAGA (SEQ IDNO: 22) as the loop sequence. Thus, the disclosure includes an shRNAcomprising or consisting of the sequence:GUCCCGCUGUGCAGUCCUCCGGUACACAAUUCAAGAGAUUGUGUACCGGAGGA CUGCACAGCGGGAC(SEQ ID NO: 25). This is also referred to herein as the hRFC2-shRNA#2sequence. SEQ ID NO 25 may also include a transcription terminationsequence, such as UUUUUU, encoded by TTTTTT.

As is well known in the art, shRNA can be introduced into target cellsusing any suitable compositions and methods. In an embodiment, the shRNAis introduced to the cell by way of a lentiviral expression system.Suitable lentiviral expression systems are known and are commerciallyavailable and can be adapted to express shRNA sequences and/or siRNAsequences disclosed herein.

In embodiments, the pharmaceutical composition comprises one or morepolynucleotides which comprise or consist of SEQ ID NO: 7, SEQ ID NO:8or SEQ ID NO:9. In embodiments, the pharmaceutical formulation isprovided in a sealed container in an article of manufacture, wherein thearticle of manufacture comprises packaging and printed material, whereinthe printed material provides an indication that the pharmaceuticalcomposition is for prophylaxis and/or therapy of cancer, such as breastcancer. The printed information can be provided on a label, or on apaper insert, or printed on the packaging material itself. The printedinformation can include information that identifies the pharmaceuticalagents (i.e., polynucleotides targeted to RFC40) in the package, andinstructions for taking or administering the pharmaceutical composition.In embodiments, the polynucleotides are provided in a pharmaceuticalformulation in one or more closed or sealed vials, bottles, or any othersuitable packaging for the sale, or distribution, or use ofpharmaceutical compositions which comprise polynucleotides for use inprophylaxis and/or therapy of cancer. In embodiments, polynucleotidesare provided in a form suitable for reconstitution into a liquidpharmaceutical composition with suitable concentrations of thepolynucleotides. In embodiments, the indication provided by the printedmaterial is an indication that the pharmaceutical composition is forprophylaxis and/or therapy of estrogen positive, or estrogen negative,or progesterone/estrogen/HER2 negative breast cancer.

In another aspect, a method is provided for prophylaxis and or therapyof cancer, including breast cancer, in a mammal comprising administeringto said mammal the above composition(s). The method may further compriseadministering an anticancer agent selected from an anti-mitotic agent,an immunomodulatory agent, and an agent targeting growth factor orestrogen receptors.

In an embodiment, the disclosure includes a method for inhibiting growthof cancer cells. The method comprises introducing into cancer cells atleast one polynucleotide, wherein the polynucleotide comprises asequence selected from any one or any combination of the sequencesdescribed herein. In embodiments, the method comprises introducing intocancer cells at least one polynucleotide which comprises, consists of,and/or encodes any one or any combination of: SEQ ID NO:3, SEQ ID NO:4,SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,ACUACGAACUGCCGUGGGUUGAAAAAUAU (SEQ ID NO:15),GUCCCGCUGUGCAGUCCUCCGGUACACAA (SEQ ID NO:16), ACUACGAACUGCCGUGGGUUG (SEQID NO:20), ACUACGAACUGCCGUGGGUUGNNNNNNNNNCAACCCACGGCAGUUCGUAGU (SEQ IDNO:21/hRFC2-shRNA#1 sequence) wherein the NNNNNNNNN may form a loopsequence in an shRNA,GUCCCGCUGUGCAGUCCUCCGGUACACAANNNNNNNNNUGUGUACCGGAGGA CUGCACAGCGGGAC (SEQID NO:24/hRFC2-shRNA#2) wherein the NNNNNNNNN may form a loop sequencein an shRNA, and wherein subsequent to its introduction, growth of thecancer cells is inhibited. In embodiments the loop sequence can be 7, 9,or 11 nucleotides in length.

One example of a suitable loop sequence comprises or consists ofUUCAAGAGA (SEQ ID NO: 22). Thus, the disclosure includes use of shRNAsequence comprising the loop sequence of SEQ ID NO: 22, such asACUACGAACUGCCGUGGGUUGUUCAAGAGACAACCCACGGCAGUUCGUAGU (SEQ ID NO:23/hRFC2-shRNA#1) andGUCCCGCUGUGCAGUCCUCCGGUACACAAUUCAAGAGAUUGUGUACCGGAGGA CUGCACAGCGGGAC(SEQ ID NO:25/hRFC2-shRNA#2 sequence.) Any shRNA sequence providedherein may also comprise a suitable transcription termination signal,such as a poly T sequence (which can be poly U, such as UUUUUU inshRNA).

In embodiments, the cancer cells are breast cancer cells. In certainaspects, the cancer cells are estrogen positive, estrogen negative orprogesterone/estrogen/HER2 negative breast cancer cells (triple-negativebreast cancer cells).

The diagnostic utility of the present invention includes in certainembodiments determining RFC40 protein amounts and/or gene amplificationand/or expression in diagnosis and prognosis of cancer, particularlybreast cancer. The disclosure provides in various embodiments methodsfor determining or prognosing/prognosticating breast cancer in anindividual comprising assessing of levels or activity of RFC40 protein,RFC40 mRNA or RFC40 gene amplification in breast tissue, whereby anindividual having breast cancer or malignancy has elevated levels oractivity of RFC40 protein, RFC40 mRNA or RFC40 gene amplification versusa normal or benign control. In one embodiment, the disclosure includes amethod for aiding in diagnosis of breast cancer in an individual. Themethod comprises testing a sample comprising breast tissue cellsobtained from the individual to determine an amount of RFC40 proteinand/or RFC40 mRNA in the sample, whereby determining increased RFC40protein and/or RFC40 mRNA relative to a non-cancer control aids in thediagnosis of breast cancer in the individual, and wherein determining anamount of RFC40 protein and/or RFC40 mRNA that is the same as thenon-cancer control indicates the individual does not have breast cancer.In embodiments, testing the sample comprises amplification of the RFC40mRNA from the sample using a polymerase chain reaction (PCR). Inembodiments, the PCR is performed using a first primer comprising thesequence ATGGAGGTGGAGGCCGTCTGTG (SEQ ID NO: 10) and second primercomprising the sequence CCTCTAGCCTGCTCACGGTGTCTTC (SEQ ID NO:11). Inembodiments, the PCR amplification is quantitative real time PCR(qRT-PCR). In embodiments, the disclosure includes fixing thedetermining of RFC40 mRNA and/or protein in a tangible medium ofexpression. In embodiments, the tangible medium of expression istransmitted or transported to a health care provider in order to aid ina diagnosis. In other embodiments, the disclosure includes determiningan increased copy number of the RFC40 gene relative to a normal,non-cancer control. In embodiments, copy number can be determined usingFISH-based approaches. In embodiments, determining an increase in RFC40mRNA and/or protein is considered indicative of the presence of anincreased copy number of the RFC40 gene in cancer cells from which theincreased mRNA and/or protein was determined.

In certain approaches, after determining an increased RFC40 proteinand/or mRNA relative to a non-cancer control, the method furthercomprising testing the sample to determine whether the breast tissuecells are estrogen positive, estrogen negative orprogesterone/estrogen/HER2 negative breast cancer cells. In embodiments,the disclosure includes determining an increased RFC40 protein and/ormRNA relative to the non-cancer control and subsequently administeringto the individual a pharmaceutical composition as described herein.

In certain embodiments, the disclosure includes RFC40 modulation,particularly inhibition, in assays to screen for specific cancer agents,particularly agents that alter cell growth, proliferation, division orprogression from G1 to S phase particularly in malignant or cancerouscells versus normal or benign cells, which is a hallmark and usefulcapability for anti-cancer compounds and agents.

Thus, another aspect of the invention provides a method for identifyinga compound that inhibits growth and/or cell division in breast cancercells, said method comprising:

-   a) contacting a test compound with an RFC40 polypeptide, fragments    or functional derivatives thereof or with a nucleic acid encoding    RFC40 or a functional derivative thereof;-   b) measuring the expression or an activity of RFC40 polypeptide; and-   c) identifying a compound capable of inhibiting the expression or    activity of said polypeptide whereby inhibition of expression or    activity of said polypeptide results in or is associated with    inhibition of growth and/or cell division in breast cancer cells.

Another aspect provides a method for identifying a compound thatinhibits growth and/or cell division in breast cancer cells, said methodcomprising:

-   a) contacting a test compound with an RFC40 polypeptide, fragments    or functional derivatives thereof or with a nucleic acid encoding    RFC40 or a functional derivative thereof;-   b) measuring the expression or an activity of said polypeptide and    identifying and/or measuring inhibition or reduction of the    expression or an activity of said polypeptide by the test compound;-   c) contacting the test compound with a population of breast cancer    cells;-   d) measuring a property related to or indicating growth or cell    division of said cells or determining the number of said cells; and-   e) identifying a compound capable of inhibiting growth and/or cell    division in breast cancer cells and demonstrating inhibition or    reduction of the expression or an activity of said polypeptide or    nucleic acid.

The above methods for identifying compounds may additionally comprisethe step of comparing the compound to be tested to a control. The RFC40polypeptide in the methods may be coupled to a detectable label. Thepolypeptide sequence in steps (a) and (b) may be performed utilizing anin vitro cell-free preparation or may be performed in a cell or cellsparticularly in mammary cells or in breast cancer cells, including celllines or primary cells.

The invention includes an assay system for screening of potential drugseffective to modulate cell division and/or proliferation of targetcancer cells, particularly breast cancer cells, by inhibiting RFC40expression or activity in the target cells. In one instance, the testdrug could be administered to a cellular sample with a cellproliferation agent or to breast cancer cells, to determine its effecton expression or activity of RFC40 in the presence of the test drug, bycomparison with a control normal or benign cell or a control in theabsence of a test drug.

In an assay, a control quantity of RFC40 or antibodies thereto, or thelike may be prepared and labeled with an enzyme, a specific bindingpartner and/or a radioactive element, and may then be introduced into acellular sample.

In the instance where a radioactive label, such as the isotopes ³H, ¹⁴C,³²P, ³⁵S, ³⁶Cl, ⁵¹Cr, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁹⁰Y, ¹²⁵I, ¹³¹I, and ¹⁸⁶Re areused, known currently available counting procedures may be utilized. Inthe instance where the label is an enzyme, detection may be accomplishedby any of the presently utilized colorimetric, spectrophotometric,fluorospectrophotometric, amperometric or gasometric techniques known inthe art.

The present invention includes an assay system which may be prepared inthe form of a test kit for the quantitative analysis of the extent ofthe presence or activity of RFC40 protein or mRNA, or to identify drugsor other agents that may mimic or block their activity. The system ortest kit may comprise a labeled component prepared by one of theradioactive and/or enzymatic techniques discussed herein, coupling alabel to the RFC40, their agonists and/or antagonists, and one or moreadditional immunochemical reagents, at least one of which is a free orimmobilized ligand, capable either of binding with the labeledcomponent, its binding partner, one of the components to be determinedor their binding partner(s).

In another aspect the disclosure includes a kit, such as an article ofmanufacture, for use aiding in diagnosis of cancer in an individual. Thekit can comprise packaging and at least one sealed container whichcontains a first primer comprising the sequence ATGGAGGTGGAGGCCGTCTGTG(SEQ ID NO:10) and second primer comprising the sequenceCCTCTAGCCTGCTCACGGTGTCTTC (SEQ ID NO:11), the packaging furthercomprising printed material, the printed material providing anindication that the first and second primers are for use in polymerasechain reaction amplification of RFC40 mRNA into a cDNA, wherein theamount of RFC40 mRNA is diagnostic of the presence or absence of thecancer. The printed material that is part of the kit can provide anindication that the amount of RFC40 mRNA is diagnostic of the presenceor absence of breast cancer. The kit can also include at least oneadditional container, the additional container comprising at least onebuffer for use in polymerase chain reaction amplification of the RFC40mRNA.

Other objects and advantages will become apparent to those skilled inthe art from a review of the following description which proceeds withreference to the following illustrative drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B (A) is a schematic representation of overexpression ofRFC40 protein and of RFC40 message in breast cancers, including inestrogen positive and estrogen negative and progesterone/estrogen/HER2negative breast cancers; (B) provides the amino acid sequence of humanRFC40 (RFC2) (Accession/UniProtKB/Swiss-Prot: P35250.3) (SEQ ID NO:1).

FIGS. 2A and 2B Western blot analyses of RFC40 using MCF10A (A and B),MCF7 (A) MDA-MB-231 (A) and MDA-MB-468 (B) cell lysates. β-Actin wasused as loading control. Each well contains 35 μg of total proteinlysate.

FIG. 3 Schematic representation of 96 cores patient breast cancer tissuearrays (BTMAs) containing 12 normal, reactive and benign tumors of thebreast (pink) and 36 breast cancer samples (purple/blue) in duplicates.

FIG. 4 (A) A 96 cores patient BTMAs containing 12 normal and 36 breastcancer samples in duplicates were subjected to immunohistochemicalanalyses using polyclonal anti-RFC40 followed by incubation withHRP-conjugated secondary antibodies for 1 h. Images of the stainedsections were collected using Dako Cytomation system. (B) Graphrepresents the RFC40 staining scores (intensity of staining×percentageof cells stained) in normal, estrogen positive (ER+ve) and estrogennegative (ER−ve) samples. Values are mean±SE. * indicates P<0.05 vs.Normal.

FIG. 5 MCF10A, MCF7 and MDA-MB-231 cells were lysed and 35 μg of totalprotein lysates were analyzed on 10% SDS-polyacrylamide gels. Proteinexpression of RFC40, RFC37 and RFC36 were examined by Western blotanalyses. β-Actin was used as a loading control.

FIG. 6A-6C Total RNA was extracted from MCF10A, MCF7 and MDA-MB-231cells and 50 ng of the t-RNA isolated from each of these samples (n=2)was subjected to real-time one-step-RT-PCR. (A) and (C) Assays forquantification of RFC40 and GAPDH mRNA expression were conducted on theCycler and the amplified products were visualized on 4% agarose gels atthe end of each run: (A) depicts results for MCF10A, MCF7 and MDA-MB-231cells, (C) depicts results for MCF10A and MDA-MB-468 cells. (B) Graphsrepresent the increases in the mRNA levels calculated from the crossingpoint deviation of all the samples and normalized with GAPDH values.

FIGS. 7A and 7B (A) MCF10A and MDA-MB-231 cells were fixed and subjectedto immunofluorescence microscopy using polyclonal anti-RFC40 followed byincubation with Alexa-488-conjugated secondary antibodies for 1 h.Images of the stained sections were collected using an Nikon A1microscope with Plan ×40/NA 0.25 Phi objective. (B) MCF10A, MCF7 andMDA-MB-231 cells were subjected to flow cytometric analyses using DAPI(MCF10A) and PI (MCF7 and MDA-MB-231), respectively. Histogramsrepresent the percent of cells in G1, S and G2 phases respectively.

FIG. 8 96 cores patient BTMAs containing 12 normal and 36 breast cancersamples in duplicates were subjected to immunohistochemical analysesusing polyclonal anti-RFC40 followed by incubation with HRP-conjugatedsecondary antibodies for 1 h. Images of the stained sections werecollected using Dako Cytomation system. The number of normal/benign andcancer cells with RFC40 positive nuclear staining (arrows) was measuredfor 3 fields/sample.

FIG. 9 is a schematic representation of RFC40 increased gene copynumbers in breast cancers, including in estrogen positive and estrogennegative and progesterone/estrogen/HER2 negative breast cancers.

FIG. 10 Genomic DNA isolated from Adult normal breast tissue, MCF10A andMDA-MB-468 cells was subjected to PCR to amplify a 206 bp fragment ofthe RFC40 promoter. Amplified products were analyzed on 4% agarose gel.

FIG. 11A-11B BTMAs were subjected to Fluorescent in-situ hybridization(FISH) using probe for RFC40 gene (RED; white arrows) on chromosome 7and a centromeric enumeration probe for chromosome 7 (CEP7; GREEN;yellow arrows) as an internal control. Slides were imaged with OlympusBX61 microscope. (A) represents RFC40 gene amplification and (B)represents polysomy of chromosome 7.

FIG. 12 Schematic representation of the oncogenic properties of RFC40protein. Over-expression of RFC40 in non-cancerous breast epithelialcells lead to oncogenic transformation of these cells with changes fromepithelial to stromal or mesenchymal phenotype.

FIG. 13 Western blot analyses for cGFP, RFC40 and β-actin using lysatesobtained from GFP-Ad and RFC40-Ad transfected MCF10 cells.

FIG. 14A-14C Univariate FASC analysis of cellular DNA content in GFP-Adand RFC40-Ad over-expressed MCF10A cells. A representative histogram forGFP-Ad (A) and RFC40-Ad over-expressed (B) MCF10A cells with thepercentage of cells in G1, S and G2-phases respectively, is shown. (C)Western blot analyses for Cyclin D1, Cyclin A and Cyclin B1 usinglysates obtained from GFP-Ad and RFC40-Ad transfected MCF10 cells.

FIGS. 15A and 15B Control (A) and RFC40-Ad (B) transfected MCF10A cellswere subjected to DIC microscopy using Nikon Eclipse TE2000-E (20×).Arrows indicate stromal-like phenotype in RFC40-Ad transfected MCF10Acells.

FIG. 16 Western blot analyses for E-cadherin and N-cadherin usinglysates obtained from GFP-Ad and RFC40-Ad transfected MCF10 cells.

FIGS. 17A and 17B Univariate analysis of cellular DNA content in MCF10Aand MDA-MB-468 cells: MCF10A and MDA-MB-468 cells (1×10⁶) were subjectedto flowcytometric analysis using 4′,6′-diamidino-2-phenylindole (DAPI)as described previously. A representative histogram for MCF10A (A) andMDA-MB-468 (B) is shown.

FIG. 18 provides human RFC40 mRNA nucleic acid sequence (SEQ ID NO:2)and the Smartpool siRNA RNA sequences for each of RFC40-siRNA-S1 (SEQ IDNO:3), RFC40-siRNA-S2 (SEQ ID NO:4), RFC40-siRNA-S3 (SEQ ID NO:5), andRFC40-siRNA-S4 (SEQ ID NO:6). The non-targeting siRNA sequence (NT) (SEQID NO:14) is also indicated. The RFC40 mRNA sequence is provided as thecDNA sequence.

FIG. 19A-19D MCF10A (non-cancerous; A), MCF7 (estrogen positive breastcancer cells; B), MDA-MB-231 (estrogen negative breast cancer cells; C)and MDA-MB-468 (triple negative like breast cancer cells; D) cells weretransfected with non-targeting (NT; A & D), Lamin A/C-LAC; B & C),glucose-6-phosphate-dehydrogenase (G6PD; A) and RFC40 siRNA-smartpool(100 nM; cocktail of four different sequences; A-D) as indicated in thefigure for 72 hr. Cells lysates were subjected to Western blot analysisusing anti-RFC40, anti-RIα and anti-G6PD antibodies, respectively.β-Actin was used as loading control.

FIG. 20A-20D MCF10A, MCF7, MDA-MB-231 and MDA-MB-468 cells weretransfected with Smartpool RFC40-SiRNA (100 nM) for 72 hr. The cellswere trypsinized, resuspended in 1×PBS and counted using ahemocytometer. Graph represents the number of MCF10A (A; n=4)) MCF7 (B;n=3), MDA-MB-231 (C; n=3) and MDA-MB-468 (D; n=3) cells vs theuntransfected (UT) and RFC40-siRNA treated cells.

FIG. 21A-21B MCF10A and MDA-MBA-468 cells were transfected withSmartpool RFC40-SiRNA (100 nM) for 72 hr. Cells were incubated withHoechst 33342 for 45 min at 37° C. Immunofluorescent microscopy wasperformed to determine the presence of apoptotic nuclei in theRFC40-siRNA treated MCF10A and MDA-MB-468 cells, respectively, usingNikon microscope (20× magnification). Unt=untransfected,NT=non-targeting.

FIGS. 22A and 22B MCF10A (non-cancerous; A) and MDA-MB-231 (estrogennegative breast cancer cells; B) cells were transfected with nontargeting (NT) and RFC40-S1/S2/S3/S4-siRNA (100 nM; four individualsequences) for 72 hr. Cells lysates were subjected to Western blotanalysis using anti-RFC40 antibody. β-Actin was used as loading control.

FIGS. 23A and 23B MCF10A and MDA-MB-231 cells were transfected withRFC40-SiRNA-S1/S2/S3/S4 (100 nM) for 72 hr. Cell number analyses wasperformed using Cyquant cell number analyses kit. Graphs represent thenumber of MCF10A (A; n=8) and MDA-MB-231 (B; n=8) cells vs theuntransfected (UT) and RFC40-siRNA-S1/S2/S3/S4 treated cells.

FIG. 24 depicts miR-hsa-125-3p miRNA sequence and four modified designedalternative miR sequences (#1, #2, #3 and #4) aligned with RFC40 mRNAsequence. The RFC2-mRNA-3′UTR alignment sequence shown in the 5′->3′direction is SEQ ID NO: 17 (ccgaggCAGGUGGAUCACCUGa) as the bottom strandin the alignment. The hsa-miR-125a-3p sequence and the modified miRNAsequences are shown in the 3′->5′ direction as the top strand in FIG.24, and are given in the 5′-3′ direction in the accompanying sequencelisting and the text of this description. Nucleotide changes in themodified miRNAs relative to the miR-hsa-125-3p miRNA are shown in thetext here as bold and italicized and in bold in FIG. 24. ThemiR-hsa-125-3p miRNA sequence is SEQ ID NO:7 (aCAGGUGAGGUUCUUGggagcc).The #1 modified sequence is SEQ ID NO:8 (aCAGGUGAUCCACUUGggagcc). The #2modified sequence is SEQ ID NO:9 (aCAGGUGAGGAUAACAggagcc). The #3modified sequence is SEQ ID NO:18 (aCAGGUGAGGUUCCUGggagcc). The #4modified sequence is SEQ ID NO:19 (aCAGGUGAGGCUCCUGggagcc).

FIGS. 25A and 25B MCF10A (A) and MDA-MB-231 (B) cells were transfectedwith negative control miRNA#1 and miR-hsa-125a-3p (100 nM), respectivelyfor 72 hr. Cells lysates were subjected to Western blot analysis usinganti-RFC40 antibody. β-Acin was used as loading control.

FIGS. 26A and 26B MCF10A and MDA-MB-231 cells were transfected withmiR-hsa-125a-3p (100 nM) for 72 hr. The cells were trypsinized,resuspended in 1×PBS and counted using a hemocytometer. Graph representsthe number of MCF10A (A; n=10) and MDA-MB-231 (B; n=9) cells vs theuntransfected (UT) and miR-hsa-125a-3p treated cells.

FIG. 27 provides hRFC2-shRNA sequences and illustrates (A) shRNA oligodesign; (B) hRFC2-shRNA#1 sequence (SEQ ID NO:23, comprising a definedloop sequence), and (C) hRFC2-shRNA #2 sequence (SEQ ID NO:25,comprising a defined loop sequence). The two shRNAs against the humanRFC2 gene (hRFC2-shRNA#1 and hRFC2-shRNA#2) were synthesized, sequencedand PAGE purified. The shRNAs were sub-cloned into an adenoviral shuttlevector, in between the BamHI/Bgl II multiple cloning sites, withTTCAAGAGA as the loop sequence and TTTTTT as the termination sequence.The BamHI/Bgl II sites are not transcribed and are not part of the shRNAsequences shown. The shuttle vector harbored dual promoters, with thehRFC2-shRNAs being expressed under the control of the human U6 promoteralong with a green fluorescent protein (GFP) under the CMV promoter(Ad-GFP-U6-hRFC2-shRNA#1 or 2, described further in the examples.

FIG. 28 Female athymic nude mice (CRL: NU(NCr)-Foxn1nu, Charles River)used for the in vivo studies were nine weeks old, with a body weight(BW) range of 19.5-26.0 g, at the beginning of the study. Twenty-twodays after tumor cell implantation, on day 1 of the treatment (D1),animals were sorted into three groups (n=10/group) as follows: Group 1:Ad-GFP; Group 2: Ad-GFP-U6-hRFC2-shRNA (#1); and Group 3:Ad-GFP-U6-hRFC2-shRNA (#2). The animals were monitored daily for generalobservations of appearance (i.e., grooming, posture, movement about thecage, etc.) and behavior to help track the progression of themodel/treatment. Measurements of body weight and tumor volume (bycaliper) were taken twice a week. The graph represents a summary of thedata showing percent group mean body weight changes across the threeGroups from Day 1-Day 28 (after the beginning of the treatment).Statistical analysis was performed by Two-Way ANOVA.

FIG. 29 Female athymic nude mice (CRL: NU(NCr)-Foxn1nu, Charles River)were used for the in vivo studies. MDA-MB231 cells, used forimplantation, were harvested during log phase growth and resuspended incold PBS. Each mouse was injected subcutaneously in the right flank with5×106 cells (0.1 mL cell suspension). Tumors were calipered in twodimensions to monitor growth as their mean volume approached the desired90 to 130 mm3 range. Tumor weight was estimated with the assumption that1 mg is equivalent to 1 mm3 of tumor volume. Twenty-two days after tumorcell implantation, on D1 of the treatment, animals were sorted intothree groups (n=10/group) as follows: Group 1: Ad-GFP; Group 2:Ad-GFP-U6-hRFC2-shRNA (#1); and Group 3: Ad-GFP-U6-hRFC2-shRNA (#2),with individual tumor volumes of 108 to 144 mm3, and group mean tumorvolumes of 113-115 mm3. Tumors were measured with a caliper twice weeklyfor the duration of the study. The graph represents a summary of thedata showing tumor volume distribution changes across the three Groupsfrom Day 1-Day 28 (after the beginning of the treatment or 50 days aftertumor cell implantation). Statistical Significance for Two-Way ANOVA:ns=non-significant, *=0.01<P<0.05, **=0.001<P<0.01, ***=P<0.001 whencompared to Group 1. TGI>60% indicates potential therapeutic activity.

DETAILED DESCRIPTION

In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Sambrook et al, “Molecular Cloning:A Laboratory Manual” (1989); “Current Protocols in Molecular Biology”Volumes I-III [Ausubel, R. M., ed. (1994)]; “Cell Biology: A LaboratoryHandbook” Volumes I-III [J. E. Celis, ed. (1994))]; “Current Protocolsin Immunology” Volumes I-III [Coligan, J. E., ed. (1994)];“Oligonucleotide Synthesis” (M. J. Gait ed. 1984); “Nucleic AcidHybridization” [B. D. Hames & S. J. Higgins eds. (1985)]; “TranscriptionAnd Translation” [B. D. Hames & S. J. Higgins, eds. (1984)]; “AnimalCell Culture” [R. I. Freshney, ed. (1986)]; “Immobilized Cells AndEnzymes” [IRL Press, (1986)]; B. Perbal, “A Practical Guide To MolecularCloning” (1984).

Therefore, if appearing herein, the following terms shall have thedefinitions set out below.

The terms “RFC40, “RFC2”, “Replication Factor C 40” and “ReplicationFactor C 40 kD protein” and any variants not specifically listed, may beused herein interchangeably, and as used throughout the presentapplication and claims refer to proteinaceous material including singleor multiple proteins, and extends to those proteins having the aminoacid sequence data described herein and presented in FIG. 1 (SEQ IDNO:1) and the encoding nucleic acid sequence in FIG. 18 (SEQ ID NO:2),and the profile of activities set forth herein and in the Claims.Accordingly, proteins displaying substantially equivalent or alteredactivity are likewise contemplated. These modifications may bedeliberate, for example, such as modifications obtained throughsite-directed mutagenesis, or may be accidental, such as those obtainedthrough mutations in hosts that are producers of the complex or itsnamed subunits. Also, the terms “RFC40, “RFC2”, “Replication Factor C40” and “Replication Factor C 40 kD protein” are intended to includewithin their scope proteins specifically recited herein as well as allsubstantially homologous analogs and allelic variations.

The amino acid residues described herein are preferred to be in the “L”isomeric form. However, residues in the “D” isomeric form can besubstituted for any L-amino acid residue, as long as the desiredfunctional property of immunoglobulin-binding is retained by thepolypeptide. NH₂ refers to the free amino group present at the aminoterminus of a polypeptide. COOH refers to the free carboxy group presentat the carboxy terminus of a polypeptide. In keeping with standardpolypeptide nomenclature, J. Biol. Chem., 243:3552-59 (1969),abbreviations for amino acid residues are shown in the following Tableof Correspondence:

TABLE OF CORRESPONDENCE SYMBOL 1-Letter 3-Letter AMINO ACID Y Tyrtyrosine G Gly glycine F Phe phenylalanine M Met methionine A Alaalanine S Ser serine I Ile isoleucine L Leu leucine T Thr threonine VVal valine P Pro proline K Lys lysine H His histidine Q Gln glutamine EGlu glutamic acid W Trp tryptophan R Arg arginine D Asp aspartic acid NAsn asparagine C Cys cysteine

It should be noted that all amino-acid residue sequences are representedherein by formulae whose left and right orientation is in theconventional direction of amino-terminus to carboxy-terminus.Furthermore, it should be noted that a dash at the beginning or end ofan amino acid residue sequence indicates a peptide bond to a furthersequence of one or more amino-acid residues. The above Table ispresented to correlate the three-letter and one-letter notations whichmay appear alternately herein.

A “replicon” is any genetic element (e.g., plasmid, chromosome, virus)that functions as an autonomous unit of DNA replication in vivo; i.e.,capable of replication under its own control.

A “vector” is a replicon, such as plasmid, phage or cosmid, to whichanother DNA segment may be attached so as to bring about the replicationof the attached segment.

A “DNA molecule” refers to the polymeric form of deoxyribonucleotides(adenine, guanine, thymine, or cytosine) in its either single strandedform, or a double-stranded helix. This term refers only to the primaryand secondary structure of the molecule, and does not limit it to anyparticular tertiary forms. Thus, this term includes double-stranded DNAfound, inter alia, in linear DNA molecules (e.g., restrictionfragments), viruses, plasmids, and chromosomes. In discussing thestructure of particular double-stranded DNA molecules, sequences may bedescribed herein according to the normal convention of giving only thesequence in the 5′ to 3′ direction along the nontranscribed strand ofDNA (i.e., the strand having a sequence homologous to the mRNA).

Transcriptional and translational control sequences are DNA regulatorysequences, such as promoters, enhancers, polyadenylation signals,terminators, and the like, that provide for the expression of a codingsequence in a host cell.

A “promoter sequence” is a DNA regulatory region capable of binding RNApolymerase in a cell and initiating transcription of a downstream (3′direction) coding sequence. For purposes of defining the presentinvention, the promoter sequence is bounded at its 3′ terminus by thetranscription initiation site and extends upstream (5′ direction) toinclude the minimum number of bases or elements necessary to initiatetranscription at levels detectable above background. Within the promotersequence will be found a transcription initiation site (convenientlydefined by mapping with nuclease S1), as well as protein binding domains(consensus sequences) responsible for the binding of RNA polymerase.Eukaryotic promoters will often, but not always, contain “TATA” boxesand “CAT” boxes. Prokaryotic promoters contain Shine-Dalgarno sequencesin addition to the −10 and -35 consensus sequences.

An “expression control sequence” is a DNA sequence that controls andregulates the transcription and translation of another DNA sequence. Acoding sequence is “under the control” of transcriptional andtranslational control sequences in a cell when RNA polymerasetranscribes the coding sequence into mRNA, which is then translated intothe protein encoded by the coding sequence.

A “signal sequence” can be included before the coding sequence. Thissequence encodes a signal peptide, N-terminal to the polypeptide, thatcommunicates to the host cell to direct the polypeptide to the cellsurface or secrete the polypeptide into the media, and this signalpeptide is clipped off by the host cell before the protein leaves thecell. Signal sequences can be found associated with a variety ofproteins native to prokaryotes and eukaryotes.

The term “oligonucleotide,” as used herein in referring to the probe ofthe present invention, is defined as a molecule comprised of two or moreribonucleotides, preferably more than three. Its exact size will dependupon many factors which, in turn, depend upon the ultimate function anduse of the oligonucleotide.

The term “primer” as used herein refers to an oligonucleotide, whetheroccurring naturally as in a purified restriction digest or producedsynthetically, which is capable of acting as a point of initiation ofsynthesis when placed under conditions in which synthesis of a primerextension product, which is complementary to a nucleic acid strand, isinduced, i.e., in the presence of nucleotides and an inducing agent suchas a DNA polymerase and at a suitable temperature and pH. The primer maybe either single-stranded or double-stranded and must be sufficientlylong to prime the synthesis of the desired extension product in thepresence of the inducing agent. The exact length of the primer willdepend upon many factors, including temperature, source of primer anduse of the method. For example, for diagnostic applications, dependingon the complexity of the target sequence, the oligonucleotide primertypically contains 15-25 or more nucleotides, although it may containfewer nucleotides.

Primers are selected to be “substantially” complementary to differentstrands of a particular target DNA sequence. This means that the primersmust be sufficiently complementary to hybridize with their respectivestrands. Therefore, the primer sequence need not reflect the exactsequence of the template. For example, a non-complementary nucleotidefragment may be attached to the 5′ end of the primer, with the remainderof the primer sequence being complementary to the strand. Alternatively,non-complementary bases or longer sequences can be interspersed into theprimer, provided that the primer sequence has sufficient complementaritywith the sequence of the strand to hybridize therewith and thereby formthe template for the synthesis of the extension product.

As used herein, the terms “restriction endonucleases” and “restrictionenzymes” refer to bacterial enzymes, each of which cut double-strandedDNA at or near a specific nucleotide sequence.

A cell has been “transformed” by exogenous or heterologous DNA when suchDNA has been introduced inside the cell. The transforming DNA may or maynot be integrated (covalently linked) into chromosomal DNA making up thegenome of the cell. In prokaryotes, yeast, and mammalian cells forexample, the transforming DNA may be maintained on an episomal elementsuch as a plasmid. With respect to eukaryotic cells, a stablytransformed cell is one in which the transforming DNA has becomeintegrated into a chromosome so that it is inherited by daughter cellsthrough chromosome replication. This stability is demonstrated by theability of the eukaryotic cell to establish cell lines or clonescomprised of a population of daughter cells containing the transformingDNA. A “clone” is a population of cells derived from a single cell orcommon ancestor by mitosis. A “cell line” is a clone of a primary cellthat is capable of stable growth in vitro for many generations.

Two DNA sequences are “substantially homologous” when at least about 75%(preferably at least about 80%, and most preferably at least about 90 or95%) of the nucleotides match over the defined length of the DNAsequences. Sequences that are substantially homologous can be identifiedby comparing the sequences using standard software available in sequencedata banks, or in a Southern hybridization experiment under, forexample, stringent conditions as defined for that particular system.Defining appropriate hybridization conditions is within the skill of theart. See, e.g., Maniatis et al., supra; DNA Cloning, Vols. I & II,supra; Nucleic Acid Hybridization, supra.

It should be appreciated that also within the scope of the presentinvention are DNA sequences encoding RFC40 which code for a polypeptidehaving the same amino acid sequence as provided in FIG. 1 (SEQ ID NO:1),but which are degenerate to the sequence of FIG. 18 (SEQ ID NO:2). By“degenerate to” is meant that a different three-letter codon is used tospecify a particular amino acid. It is well known in the art that thefollowing codons can be used interchangeably to code for each specificamino acid:

Phenylalanine (Phe or F) UUU or UUC Leucine (Leu or L)UUA or UUG or CUU or CUC or CUA or CUG Isoleucine (Ile or I)AUU or AUC or AUA Methionine (Met or M) AUG Valine (Val or V)GUU or GUC of GUA or GUG Serine (Ser or S)UCU or UCC or UCA or UCG or AGU or AGC Proline (Pro or P)CCU or CCC or CCA or CCG Threonine (Thr or T) ACU or ACC or ACA or ACGAlanine (Ala or A) GCU or GCG or GCA or GCG Tyrosine (Tyr or Y)UAU or UAC Histidine (His or H) CAU or CAC Glutamine (Gln or Q)CAA or CAG Asparagine (Asn or N) AAU or AAC Lysine (Lys or K) AAA or AAGAspartic Acid (Asp or D) GAU or GAC Glutamic Acid (Glu or E) GAA or GAGCysteine (Cys or C) UGU or UGC Arginine (Arg or R)CGU or CGC or CGA or CGG or AGA or AGG Glycine (Gly or G)GGU or GGC or GGA or GGG Tryptophan (Trp or W) UGG Termination codonUAA (ochre) or UAG (amber) or UGA (opal)

It should be understood that the codons specified above are for RNAsequences. The corresponding codons for DNA have a T substituted for U.

Mutations can be made in RFC40 sequence as set out in FIG. 18 (SEQ IDNO:2) such that a particular codon is changed to a codon which codes fora different amino acid. Such a mutation is generally made by making thefewest nucleotide changes possible. A substitution mutation of this sortcan be made to change an amino acid in the resulting protein in anon-conservative manner (i.e., by changing the codon from an amino acidbelonging to a grouping of amino acids having a particular size orcharacteristic to an amino acid belonging to another grouping) or in aconservative manner (i.e., by changing the codon from an amino acidbelonging to a grouping of amino acids having a particular size orcharacteristic to an amino acid belonging to the same grouping). Such aconservative change generally leads to less change in the structure andfunction of the resulting protein. A non-conservative change is morelikely to alter the structure, activity or function of the resultingprotein. The present invention should be considered to include sequencescontaining conservative changes which do not significantly alter theactivity or binding characteristics of the resulting protein.

The following is one example of various groupings of amino acids:

Amino acids with nonpolar R groups: Alanine, Valine, Leucine,Isoleucine, Proline, Phenylalanine, Tryptophan, Methionine

Amino acids with uncharged polar R groups: Glycine, Serine, Threonine,Cysteine, Tyrosine, Asparagine, Glutamine

Amino acids with charged polar R groups (negatively charged at Ph 6.0):Aspartic acid, Glutamic acid

Basic amino acids (positively charged at pH 6.0): Lysine, Arginine,Histidine (at pH 6.0)

Another grouping may be those amino acids with phenyl groups:Phenylalanine, Tryptophan, Tyrosine

Another grouping may be according to molecular weight (i.e., size of Rgroups):

Glycine 75 Alanine 89 Serine 105 Proline 115 Valine 117 Threonine 119Cysteine 121 Leucine 131 Isoleucine 131 Asparagine 132 Aspartic acid 133Glutamine 146 Lysine 146 Glutamic acid 147 Methionine 149 Histidine (atpH 6.0) 155 Phenylalanine 165 Arginine 174 Tyrosine 181 Tryptophan 204

Particularly preferred substitutions are:

-   -   Lys for Arg and vice versa such that a positive charge may be        maintained;    -   Glu for Asp and vice versa such that a negative charge may be        maintained;    -   Ser for Thr such that a free —OH can be maintained; and    -   Gln for Asn such that a free NH₂ can be maintained.

Amino acid substitutions may also be introduced to substitute an aminoacid with a particularly preferable property. For example, a Cys may beintroduced a potential site for disulfide bridges with another Cys. AHis may be introduced as a particularly “catalytic” site (i.e., His canact as an acid or base and is the most common amino acid in biochemicalcatalysis). Pro may be introduced because of its particularly planarstructure, which induces β-turns in the protein's structure.

Two amino acid sequences are “substantially homologous” when at leastabout 70% of the amino acid residues (preferably at least about 80%, andmost preferably at least about 90 or 95%) are identical, or representconservative substitutions.

A “heterologous” region of the DNA construct is an identifiable segmentof DNA within a larger DNA molecule that is not found in associationwith the larger molecule in nature. Thus, when the heterologous regionencodes a mammalian gene, the gene will usually be flanked by DNA thatdoes not flank the mammalian genomic DNA in the genome of the sourceorganism. Another example of a heterologous coding sequence is aconstruct where the coding sequence itself is not found in nature (e.g.,a cDNA where the genomic coding sequence contains introns, or syntheticsequences having codons different than the native gene). Allelicvariations or naturally-occurring mutational events do not give rise toa heterologous region of DNA as defined herein.

An “antibody” is any immunoglobulin, including antibodies and fragmentsthereof, that binds a specific epitope. The term encompasses polyclonal,monoclonal, and chimeric antibodies, the last mentioned described infurther detail in U.S. Pat. Nos. 4,816,397 and 4,816,567.

An “antibody combining site” is that structural portion of an antibodymolecule comprised of heavy and light chain variable and hypervariableregions that specifically binds antigen.

The phrase “antibody molecule” in its various grammatical forms as usedherein contemplates both an intact immunoglobulin molecule and animmunologically active portion of an immunoglobulin molecule.

Exemplary antibody molecules are intact immunoglobulin molecules,substantially intact immunoglobulin molecules and those portions of animmunoglobulin molecule that contains the paratope, including thoseportions known in the art as Fab, Fab′, F(ab′)₂ and F(v), which portionsare preferred for use in the therapeutic methods described herein.

Fab and F(ab′)₂ portions of antibody molecules are prepared by theproteolytic reaction of papain and pepsin, respectively, onsubstantially intact antibody molecules by methods that are well-known.See for example, U.S. Pat. No. 4,342,566 to Theofilopolous et al. Fab′antibody molecule portions are also well-known and are produced fromF(ab′)₂ portions followed by reduction of the disulfide bonds linkingthe two heavy chain portions as with mercaptoethanol, and followed byalkylation of the resulting protein mercaptan with a reagent such asiodoacetamide. An antibody containing intact antibody molecules ispreferred herein.

The phrase “monoclonal antibody” in its various grammatical forms refersto an antibody having only one species of antibody combining sitecapable of immunoreacting with a particular antigen. A monoclonalantibody thus typically displays a single binding affinity for anyantigen with which it immunoreacts. A monoclonal antibody may thereforecontain an antibody molecule having a plurality of antibody combiningsites, each immunospecific for a different antigen; e.g., a bispecific(chimeric) monoclonal antibody.

The phrase “pharmaceutically acceptable” refers to molecular entitiesand compositions that are physiologically tolerable and do not typicallyproduce an allergic or similar untoward reaction, such as gastric upset,dizziness and the like, when administered to a human.

The phrase “therapeutically effective amount” is used herein to mean anamount sufficient to prevent, and preferably reduce by at least about 30percent, more preferably by at least 50 percent, most preferably by atleast 90 percent, a clinically significant change in the S phaseactivity of a target cellular mass, or other feature of pathology suchas for example, elevated blood pressure, fever or white cell count asmay attend its presence and activity.

A DNA sequence is “operatively linked” to an expression control sequencewhen the expression control sequence controls and regulates thetranscription and translation of that DNA sequence. The term“operatively linked” includes having an appropriate start signal (e.g.,ATG) in front of the DNA sequence to be expressed and maintaining thecorrect reading frame to permit expression of the DNA sequence under thecontrol of the expression control sequence and production of the desiredproduct encoded by the DNA sequence. If a gene that one desires toinsert into a recombinant DNA molecule does not contain an appropriatestart signal, such a start signal can be inserted in front of the gene.

The term “standard hybridization conditions” refers to salt andtemperature conditions substantially equivalent to 5×SSC and 65° C. forboth hybridization and wash. However, one skilled in the art willappreciate that such “standard hybridization conditions” are dependenton particular conditions including the concentration of sodium andmagnesium in the buffer, nucleotide sequence length and concentration,percent mismatch, percent formamide, and the like. Also important in thedetermination of “standard hybridization conditions” is whether the twosequences hybridizing are RNA-RNA, DNA-DNA or RNA-DNA. Such standardhybridization conditions are easily determined by one skilled in the artaccording to well known formulae, wherein hybridization is typically10-20^(N)C below the predicted or determined T_(m) with washes of higherstringency, if desired.

In one aspect, the present invention identifies and validates a DNAreplication protein, RFC40, as a non-receptor based molecular marker andspecific target for breast cancer, irrespective or its receptor status,including TNBC. The studies provided herein establish that RFC40 proteinand message, as well as RFC40 gene copy numbers, are increased in breastcancers, including in estrogen sensitive, estrogen resistant and triplenegative breast cancer (TNBC). The present studies demonstrate thatinhibition or modulation of RFC40 such that its activity or expressionis reduced or blocked results in reduction in cell numbers andinhibition of cell proliferation or division in estrogen positive (ERpositive), estrogen negative (ER negative), and progesterone, estrogenand human epidermal growth factor receptor 2-HER2 negative or triplenegative breast cancer (TNBC) cells.

Thus, RFC40 provides a non-receptor diagnostic and prognostic marker anda therapeutic or interventional target involved in cell division andproliferation which is independent of growth factor and endocrinereceptor status. Inhibition or modulation of RFC40 is thereforeapplicable in early stage breast cancer, late stage breast cancer, ondrug failure or resistance to receptor-based therapies, and in instancesof recurrence.

Further, the studies herein establish that RFC40 can be specificallyinhibited in cancer cells, without effects in concomitant normal orbenign cells, thereby providing RFC40 as a specific anti-cancer agentwith onco-specificity in rapidly dividing and proliferating cancercells, without toxic or unintended effects in normal cells.

The possibilities both diagnostic and therapeutic that are raised inpart by the recognition of RFC40 gene amplification and increasedmessage and protein in cancer cells, particularly in breast cancer cellsand irrespective of growth factor or estrogen receptor status. Thepresent invention contemplates pharmaceutical intervention in theexpression, activity or necessary protein interactions of RFC40 in acancer cell, to modulate the cell division and proliferation, includingparticularly the G1 to S phase transition.

Thus, in instances where it is desired to reduce or inhibit cancer,including but not necessarily limited to breast cancer, cell division orproliferation, agents or compounds may be introduced to blockRFC40-mediated function or activity in a cancer cell, including theexpression of RFC40, the activity of RFC40 protein, or the interactionof RFC40 with those factors causally connected with its activity ornecessary nuclear localization.

RFC40 inhibitors or agents modulating RFC40-mediated cellular effectsmay be prepared in pharmaceutical compositions, with a suitable carrierand at a strength effective for administration by various means to apatient experiencing an adverse medical condition associated withspecific enhanced expression or activity of RFC40, or inhyper-proliferative diseases, such as cancer, including breast cancer,for the treatment thereof. A variety of administrative techniques may beutilized, among them parental techniques such as subcutaneous,intravenous and intra-peritoneal injections, catheterizations and thelike, oral administration, and dermal applications. Average quantitiesof the RFC40 inhibitors or agents may vary and in particular should bebased upon the recommendations and prescription of a qualified physicianor veterinarian.

Also, antibodies directed against RFC40, including both polyclonal andmonoclonal antibodies, and drugs that modulate the production oractivity of RFC40 or that bind with affinity to RFC40 protein, messageor nucleic acid, and/or their subunits may possess certain diagnosticapplications and may for example, be utilized for the purpose ofdetecting and/or measuring conditions such as cancer, including breastcancer.

Methods for producing polyclonal anti-polypeptide antibodies arewell-known in the art. A monoclonal antibody, typically containing Faband/or F(ab′)₂ portions of useful antibody molecules, can be preparedusing the hybridoma technology described in Antibodies—A LaboratoryManual, Harlow and Lane, eds., Cold Spring Harbor Laboratory, New York(1988), which is incorporated herein by reference. The generalmethodology for making monoclonal antibodies by hybridomas is wellknown. Immortal, antibody-producing cell lines can also be created bytechniques other than fusion, such as direct transformation of Blymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus.See, e.g., M. Schreier et al., “Hybridoma Techniques” (1980); Hammerlinget al., “Monoclonal Antibodies And T-cell Hybridomas” (1981); Kennett etal., “Monoclonal Antibodies” (1980); see also U.S. Pat. Nos. 4,341,761;4,399,121; 4,427,783; 4,444,887; 4,451,570; 4,466,917; 4,472,500;4,491,632; 4,493,890.

Panels of monoclonal antibodies produced against RFC40 peptides can bescreened for various properties; i.e., isotype, epitope, affinity, etc.Of particular interest are monoclonal antibodies that neutralize theactivity of the RFC40 or its subunits. Such monoclonals can be readilyidentified in RFC40 activity assays.

Preferably, the anti-RFC40 antibody used in the diagnostic methods ofthis invention is an affinity purified polyclonal antibody. Morepreferably, the antibody is a monoclonal antibody (mAb). In addition, itis preferable for the anti-RFC40 antibody molecules used herein be inthe form of Fab, Fab′, F(ab′)₂ or F(v) portions of whole antibodymolecules.

As suggested earlier, the diagnostic method of the present inventioncomprises examining a cellular sample or medium by means of an assayincluding an effective amount of an antagonist to a ˜/protein, such asan anti-˜antibody, preferably an affinity-purified polyclonal antibody,and more preferably a mAb. In addition, it is preferable for theanti-˜antibody molecules used herein be in the form of Fab, Fab′,F(ab′)₂ or F(v) portions or whole antibody molecules. As previouslydiscussed, patients capable of benefiting from this method include thosesuffering from cancer, a pre-cancerous lesion, a viral infection orother like pathological derangement. Methods for isolating the ˜ andinducing anti-˜antibodies and for determining and optimizing the abilityof anti-˜antibodies to assist in the examination of the target cells areall well-known in the art.

The present invention further contemplates pharmaceutical andtherapeutic compositions useful in practicing the therapeutic methods ofthis invention. A subject pharmaceutical or therapeutic compositionincludes, in admixture, a pharmaceutically acceptable excipient(carrier) and one or more of an RFC40 inhibitory agent or compound asdescribed herein as an active ingredient.

The preparation of therapeutic compositions which contain nucleic acidspolypeptides, analogs or active fragments, chemical agents, organic orinorganic compounds, etc as active ingredients is well understood in theart. Such compositions may be prepared as injectables, either as liquidsolutions or suspensions. Solid forms suitable for solution in, orsuspension in, liquid prior to injection can also be prepared. Thepreparation can also be emulsified. The active therapeutic ingredient isoften mixed with excipients which are pharmaceutically acceptable andcompatible with the active ingredient. Suitable excipients are, forexample, water, saline, dextrose, glycerol, ethanol, or the like andcombinations thereof. In addition, if desired, the composition cancontain minor amounts of auxiliary substances such as wetting oremulsifying agents, pH buffering agents which enhance the effectivenessof the active ingredient.

A nucleic acid, polypeptide, analog or active fragment or other compoundor agent can be formulated into the pharmaceutical or therapeuticcomposition as neutralized pharmaceutically acceptable salt forms.Pharmaceutically acceptable salts include the acid addition salts(formed with the free amino groups of the polypeptide or antibodymolecule) and which are formed with inorganic acids such as, forexample, hydrochloric or phosphoric acids, or such organic acids asacetic, oxalic, tartaric, mandelic, and the like. Salts formed from thefree carboxyl groups can also be derived from inorganic bases such as,for example, sodium, potassium, ammonium, calcium, or ferric hydroxides,and such organic bases as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, and the like.

The therapeutic RFC40 inhibitory agent or compound containingcompositions are conventionally administered intravenously, as byinjection of a unit dose, for example. The term “unit dose” when used inreference to a therapeutic composition of the present invention refersto physically discrete units suitable as unitary dosage for humans, eachunit containing a predetermined quantity of active material calculatedto produce the desired therapeutic effect in association with therequired diluent; i.e., carrier, or vehicle.

The compositions are administered in a manner compatible with the dosageformulation, and in a therapeutically effective amount. The quantity tobe administered depends on the subject to be treated, capacity of thesubject's cellular or immune system to utilize the active ingredient,and degree of inhibition of RFC40 activity or expression or of celldivision or proliferation desired. Precise amounts of active ingredientrequired to be administered depend on the judgment of the practitionerand are peculiar to each individual. However, suitable dosages may rangefrom about 0.1 to 20, preferably about 0.5 to about 10, and morepreferably one to several, milligrams of active ingredient per kilogrambody weight of individual per day and depend on the route ofadministration. Suitable regimes for initial administration andsubsequent administration are also variable, but are typified by aninitial administration followed by repeated doses at one or more hour,day, week or month intervals by a subsequent administration.Alternatively, continuous (e.g. intravenous) infusion sufficient tomaintain appropriate and sufficient concentrations in the blood or atthe cancer site cellular environment are contemplated.

The pharmaceutical or therapeutic compositions may further include aneffective amount of the RFC40 inhibitory agent or compound, and one ormore of the following active ingredients: an anti-mitotic, animmune-modulator, a growth factor modulator, an interleukin orinterferon, a kinase inhibitor, an anti-cancer antibody such as a HER2antibody and/or an EGFR antibody, an antibiotic, a steroid.

As used herein, “pg” means picogram, “ng” means nanogram, “ug” or “μg”mean microgram, “mg” means milligram, “ul” or “μl” mean microliter, “ml”means milliliter, “l” means liter.

Another feature of this invention is the expression of the nucleic acidsequences including DNA and RNA sequences disclosed herein, including inparticular the RFC40 inhibitory agents or compounds. As is well known inthe art, nucleic acid sequences may be expressed by operatively linkingthem to an expression control sequence in an appropriate expressionvector and employing that expression vector to transform an appropriateunicellular host. Such operative linking of a nucleic acid sequence ofthis invention to an expression control sequence, of course, includes,if not already part of the DNA or RNA sequence, the provision of aninitiation codon, ATG, in the correct reading frame upstream of the DNAor RNA sequence.

A wide variety of host/expression vector combinations may be employed inexpressing the nucleic sequences of this invention. Useful expressionvectors, for example, may consist of segments of chromosomal,non-chromosomal and synthetic DNA sequences. Suitable vectors includederivatives of SV40 and known bacterial plasmids, e.g., E. coli plasmidscol E1, pCR1, pBR322, pMB9 and their derivatives, plasmids such as RP4;phage DNAS, e.g., the numerous derivatives of phage λ, e.g., NM989, andother phage DNA, e.g., M13 and filamentous single stranded phage DNA;yeast plasmids such as the 2μ plasmid or derivatives thereof; vectorsuseful in eukaryotic cells, such as vectors useful in insect ormammalian cells; vectors derived from combinations of plasmids and phageDNAs, such as plasmids that have been modified to employ phage DNA orother expression control sequences; and the like.

Any of a wide variety of expression control sequences—sequences thatcontrol the expression of a nucleic acid sequence operatively linked toit—may be used in these vectors to express the nucleic acid sequences ofthis invention. Such expression control sequences include, for example,the early or late promoters of SV40, CMV, vaccinia, polyoma oradenovirus, the lac system, the trp system, the TAC system, the TRCsystem, the LTR system, the major operator and promoter regions of phageλ, the control regions of fd coat protein, the promoter for3-phosphoglycerate kinase or other glycolytic enzymes, the promoters ofacid phosphatase (e.g., Pho5), the promoters of the yeast ∀-matingfactors, and other sequences known to control the expression of genes ofprokaryotic or eukaryotic cells or their viruses, and variouscombinations thereof.

A wide variety of unicellular host cells are also useful in expressingthe nucleic acid sequences of this invention. These hosts may includewell known eukaryotic and prokaryotic hosts, such as strains of E. coli,Pseudomonas, Bacillus, Streptomyces, fungi such as yeasts, and animalcells, such as CHO, R1.1, B-W and L-M cells, African Green Monkey kidneycells (e.g., COS 1, COS 7, BSC1, BSC40, and BMT10), insect cells (e.g.,Sf9), and human cells and plant cells in tissue culture.

One skilled in the art will be able to select the proper vectors,expression control sequences, and hosts without undue experimentation toaccomplish the desired expression without departing from the scope ofthis invention.

In an embodiment, a nucleic acid sequence provided or of use inaccordance with the present invention can be prepared syntheticallyusing methods known and available in the art, including commerciallyavailable synthesizers, laboratory bench methods and in vitro methods.

Synthetic nucleic acid sequences allow convenient construction ofanalogs or “muteins”. Alternatively, nucleic acid having or encodingmuteins can be made by site-directed mutagenesis of native genes orcDNAs, cloned or synthetic nucleic acid sequences and muteins can bemade directly using conventional polypeptide or nucleic acid synthesis.

A general method for site-specific incorporation of unnatural aminoacids into proteins is described in Christopher J. Noren, Spencer J.Anthony-Cahill, Michael C. Griffith, Peter G. Schultz, Science,244:182-188 (April 1989). This method may be used to create analogs withunnatural amino acids.

Expression of RFC40 can be inhibited by various means includingexpression inhibitory agents which are nucleic acids or nucleicacid-based and that have binding activity for RFC40 nucleic acid andthereby exert an inhibitory effect on expression, translation andthereby reduce effective activity of RFC40 in cells, particularly incancer cells, particularly breast cancer cells. Such an inhibitor of theactivity or expression of RFC40 includes and may be a small interferingRNA (siRNA), microRNA (miRNA), an antisense polynucleotide, a ribozymeor a short-hairpin RNA (shRNA), particularly wherein said inhibitorcomprises a nucleic acid sequence complementary to, or engineered from,a naturally-occurring polynucleotide sequence of about 17 to about 30contiguous nucleotides of a nucleic acid encoding RFC40 polypeptide.

miRs MicroRNAs (miRNAs) are ubiquitous regulators of biologicalprocesses involved in normal development, in differentiation and indiseases, including cancer. They act by regulating gene expression atthe transcriptional and translational levels (Bartel et al (2004) Cell116:281-297). miRNAs were initially discovered by analysis of mutationscausing developmental defects in Caenorhabditis elegans (Lee R. C. et al(1993) Cell, 75, 843-854) and altered miRNA expression has been furtherdemonstrated in human cancer, including leukemia (Calin G. A. et al(2004) PNAS USA 101: 11755-11760; HayashitaY et al (2005) Cancer Res65:9628-9632; Johnson S. M. et al (2005) Cell 120:635-647; Lu J et al(2005) Nature 435:834-838; Venturini L et al (2007) Blood109:4399-4405). MicroRNAs (miRNA) regulate gene expression in a sequencespecific manner by hybridization and recruitment of multi-proteincomplexes to complementary messenger RNA (mRNA) target sequences. miRNAfunction can transiently be antagonized by antagomirs—chemicallymodified oligonucleotides complementary to individual miRNAs.

A single miRNA can target hundreds of messenger RNAs and therebymodulate protein output from their respective genes (Bartel D P (2009)Cell 136:215-233). Therefore a single or specific set of miRNAs maycontrol discrete physiological processes by regulating the production ofa few proteins that coordinate single or interrelated cellular events(e.g., cell proliferation) (Baltimore D et al (2008) Nat Immunol9:839-845; Bartel D P (2009) Cell 136:215-233).

Numerous miRNAs are known and have been identified. Known miRNAs areaccessible by name with sequence information and characteristics viapublic database(s) including the miRBase database, mirbase.org;Griffiths-Jones S (2003) Methods Mol Biol 342:129-138. Nonetheless,their specific roles in initiation and/or progression of disease(s) andtheir particular value as targets for therapies or as modulators ofdisease, including specific cancer(s) are, in many instances, stillbeing defined. Specific inhibition of one or more miRNAs can be achievedusing antagonists or antagomirs, which comprise complementary sequences,including oligonucleotides and nucleic acids, which specifically inhibitor block the expression and activity of miRNA(s). Antagomirs of miRNA(s)are provided and assessed herein, with demonstrated anti-oncogenic andanti-proliferative activity.

siRNAs. A particular inhibitory agent is a small interfering RNA (siRNA,particularly small hairpin RNA, “shRNA”). siRNA, particularly shRNA,mediate the post-transcriptional process of gene silencing by doublestranded RNA (dsRNA) that is homologous in sequence to the silenced RNA.siRNA according to the present invention comprises a sense strand of15-30, particularly 17-30, most particularly 17-25 nucleotidescomplementary or homologous to a contiguous 17-25 nucleotide sequenceselected from the group of exemplary siRNA sequences described in FIG.18, and an antisense strand of 15-30, particularly 17-30, mostparticularly 17-25, more specifically 19-21 nucleotides complementary tothe sense strand of encoding RNA, particularly complementary to encodingnucleic acid as set out in FIG. 18. In certain embodiments, siRNAcomprises sense and anti-sense strands that are 100 percentcomplementary to each other and the TARGET polynucleotide sequence. Inembodiments the siRNA further comprises a loop region linking the senseand the antisense strand.

A self-complementing single stranded shRNA molecule polynucleotideaccording to the present invention comprises a sense portion and anantisense portion connected by a loop region linker. Particularly, theloop region sequence is 4-30 nucleotides long, more particularly 5-15nucleotides long and most particularly 8 or 12 nucleotides long.Self-complementary single stranded siRNAs form hairpin loops and aremore stable than ordinary dsRNA. In addition, they are more easilyproduced from vectors.

Analogous to antisense RNA, the siRNA can be modified to confirmresistance to nucleolytic degradation, or to enhance activity, or toenhance cellular distribution, or to enhance cellular uptake, suchmodifications may consist of modified internucleoside linkages, modifiednucleic acid bases, modified sugars and/or chemical linkage the siRNA toone or more moieties or conjugates. The nucleotide sequences areselected according to siRNA designing rules that give an improvedreduction of the TARGET sequences compared to nucleotide sequences thatdo not comply with these siRNA designing rules (For a discussion ofthese rules and examples of the preparation of siRNA, WO 2004/094636 andUS 2003/0198627, are hereby incorporated by reference).

The present invention extends to antisense oligonucleotides andribozymes that may be used to interfere with the expression of the RFC40at the translational level. This approach utilizes antisense nucleicacid and ribozymes to block translation of a specific mRNA, either bymasking that mRNA with an antisense nucleic acid or cleaving it with aribozyme. Antisense nucleic acids are DNA or RNA molecules that arecomplementary to at least a portion of a specific mRNA molecule. (SeeWeintraub, 1990; Marcus-Sekura, 1988.) In the cell, they hybridize tothat mRNA, forming a double stranded molecule. The cell does nottranslate an mRNA in this double-stranded form. Therefore, antisensenucleic acids interfere with the expression of mRNA into protein.Oligomers of about fifteen nucleotides and molecules that hybridize tothe AUG initiation codon will be particularly efficient, since they areeasy to synthesize and are likely to pose fewer problems than largermolecules when introducing them into RFC40-producing cells. Antisensemethods have been used to inhibit the expression of many genes in vitro(Marcus-Sekura, 1988; Hambor et al., 1988).

Ribozymes are RNA molecules possessing the ability to specificallycleave other single stranded RNA molecules in a manner somewhatanalogous to DNA restriction endonucleases. Because they aresequence-specific, only mRNAs with particular sequences are inactivated.Ribozymes are catalytic RNA molecules (RNA enzymes) that have separatecatalytic and substrate binding domains. The substrate binding sequencecombines by nucleotide complementarity and, possibly, non-hydrogen bondinteractions with its target sequence. The catalytic portion cleaves thetarget RNA at a specific site. The substrate domain of a ribozyme can beengineered to direct it to a specified mRNA sequence. The ribozymerecognizes and then binds a target mRNA through complementary basepairing. Once it is bound to the correct target site, the ribozyme actsenzymatically to cut the target mRNA. Cleavage of the mRNA by a ribozymedestroys its ability to direct synthesis of the correspondingpolypeptide. Once the ribozyme has cleaved its target sequence, it isreleased and can repeatedly bind and cleave at other mRNAs.

Exemplary ribozyme forms include a hammerhead motif, a hairpin motif, ahepatitis delta virus, group I intron or RNaseP RNA (in association withan RNA guide sequence) motif or Neurospora VS RNA motif. Ribozymespossessing a hammerhead or hairpin structure are readily prepared sincethese catalytic RNA molecules can be expressed within cells fromeukaryotic promoters (Chen, et al. (1992) Nucleic Acids Res. 20:4581-9).A ribozyme of the present invention can be expressed in eukaryotic cellsfrom the appropriate DNA vector. If desired, the activity of theribozyme may be augmented by its release from the primary transcript bya second ribozyme (Ventura, et al. (1993) Nucleic Acids Res.21:3249-55).

Ribozymes may be chemically synthesized by combining anoligodeoxyribonucleotide with a ribozyme catalytic domain (20nucleotides) flanked by sequences that hybridize to the TARGET mRNAafter transcription. The oligodeoxyribonucleotide is amplified by usingthe substrate binding sequences as primers. The amplification product iscloned into a eukaryotic expression vector.

Ribozymes are expressed from transcription units inserted into DNA, RNA,or viral vectors. Transcription of the ribozyme sequences are drivenfrom a promoter for eukaryotic RNA polymerase I (pol (I), RNA polymeraseII (pol II), or RNA polymerase III (pol III). Transcripts from pol II orpol III promoters will be expressed at high levels in all cells; thelevels of a given pol II promoter in a given cell type will depend onnearby gene regulatory sequences. Prokaryotic RNA polymerase promotersare also used, providing that the prokaryotic RNA polymerase enzyme isexpressed in the appropriate cells (Gao and Huang, (1993) Nucleic AcidsRes. 21:2867-72). It has been demonstrated that ribozymes expressed fromthese promoters can function in mammalian cells (Kashani-Sabet, et al.(1992) Antisense Res. Dev. 2:3-15).

The antisense nucleic acids, siRNAs and miRs are particularlyoligonucleotides and may consist entirely of ribonucleotides, modifiedribonucleotides, deoxyribo-nucleotides, modified deoxyribonucleotides,or some combination of both. The nucleic acids can be syntheticoligonucleotides. The nucleic acids and oligonucleotides may bechemically modified, if desired, to improve stability and/orselectivity. Specific examples of some particular oligonucleotidesenvisioned for this invention include those containing modifiedbackbones, for example, phosphorothioates, phosphotriesters, methylphosphonates, short chain alkyl or cycloalkyl intersugar linkages orshort chain heteroatomic or heterocyclic intersugar linkages. Sinceoligonucleotides are susceptible to degradation by intracellularnucleases, the modifications can include, for example, the use of asulfur group to replace the free oxygen of the phosphodiester bond. Thismodification is called a phosphorothioate linkage. Phosphorothioateantisense oligonucleotides are water soluble, polyanionic, and resistantto endogenous nucleases. In addition, when a phosphorothioate antisenseoligonucleotide hybridizes to its target (for example RFC40) site, theRNA-DNA duplex activates the endogenous enzyme ribonuclease (RNase) H,which cleaves the mRNA component of the hybrid molecule.Oligonucleotides may also contain one or more substituted sugarmoieties. Particular oligonucleotides comprise one of the following atthe 2′ position: OH, SH, SCH3, F, OCN, heterocycloalkyl;heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl;an RNA cleaving group; a reporter group; an intercalator; a group forimproving the pharmacokinetic properties of an oligonucleotide; or agroup for improving the pharmacodynamic properties of an oligonucleotideand other substituents having similar properties. Similar modificationsmay also be made at other positions on the oligonucleotide, particularlythe 3′ position of the sugar on the 3′ terminal nucleotide and the 5′position of 5′ terminal nucleotide.

In addition, antisense oligonucleotides with phosphoramidite andpolyamide (peptide) linkages can be synthesized. These molecules shouldbe very resistant to nuclease degradation. Furthermore, chemical groupscan be added to the 2′ carbon of the sugar moiety and the 5 carbon (C-5)of pyrimidines to enhance stability and facilitate the binding of theantisense oligonucleotide to its TARGET site. Modifications may include2′-deoxy, O-pentoxy, O-propoxy, O-methoxy, fluoro, methoxyethoxyphosphorothioates, modified bases, as well as other modifications knownto those of skill in the art.

The DNA sequences described herein may thus be used to prepare antisensemolecules against, and ribozymes that cleave mRNAs for RFC40 and theirligands.

Another aspect of the present invention relates to a method foridentifying a compound that inhibits or reduces cell division orproliferation in cancer cells, particularly breast cancer. The methodmay comprise contacting mammalian cells with an expression-inhibitingagent that inhibits the translation in the cell of a polyribonucleotideencoding a RFC40 polypeptide. A particular embodiment relates to acomposition comprising a polynucleotide including at least one antisensestrand that functions to pair the agent with the target RFC40 mRNA, andthereby down-regulate or block the expression of target RFC40polypeptide. The inhibitory agent particularly comprises antisensepolynucleotide, a ribozyme, and a small interfering RNA (siRNA), whereinsaid agent comprises a nucleic acid sequence complementary to, orengineered from, a naturally-occurring polynucleotide sequence of RFC40,including as set out in FIG. 18 (SEQ ID NO:2).

One embodiment of the present invention relates to a method foridentifying a compound that inhibits or reduces cell division orproliferation in cancer cells, particularly breast cancer, wherein thecompound is an expression-inhibiting agent and is selected from thegroup consisting of antisense RNA, antisense oligodeoxynucleotide (ODN),a ribozyme that cleaves the polyribonucleotide coding for RFC40, a smallinterfering RNA (siRNA, particularly shRNA) that is sufficientlyhomologous to a portion of the polyribonucleotide coding for RFC40,including as set out in FIG. 18 (SEQ ID NO:2), such that the antisenseRNA, ODN, ribozyme, particularly siRNA, particularly shRNA, interfereswith the translation of the target RFC40 polyribonucleotide to thetarget RFC40 polypeptide.

Another embodiment of the present invention relates to a method foridentifying a compound for treatment of cancer, particularly breastcancer, wherein said compound is an expression-inhibiting agent such asa nucleic acid expressing the antisense RNA, antisenseoligodeoxynucleotide (ODN), a ribozyme that cleaves thepolyribonucleotide coding for RFC40, including as set out in FIG. 18(SEQ ID NO:2), a small interfering RNA (siRNA, particularly shRNA,) thatis sufficiently complementary to a portion of the polyribonucleotidecoding for RFC40, including as set out in FIG. 18 (SEQ ID NO:2), suchthat the antisense RNA, ODN, ribozyme, particularly siRNA, particularlyshRNA, interferes with the translation of the target RFC40polyribonucleotide to the target RFC40 polypeptide. Particularly theexpression-inhibiting agent is an antisense RNA, ribozyme, antisenseoligodeoxynucleotide, or siRNA, particularly shRNA, comprising apolyribonucleotide sequence that complements at least about 17 to about30 contiguous nucleotides of a nucleotide sequence coding for RFC40,including as set out in FIG. 18 (SEQ ID NO:2).

More particularly, the expression-inhibiting agent is an antisense RNA,ribozyme, antisense oligodeoxynucleotide, or siRNA, particularly shRNA,comprising a polyribonucleotide sequence that complements at least 15 toabout 30, particularly at least 17 to about 30, most particularly atleast 17 to about 25, more specifically at least 19 to about 21contiguous nucleotides of a nucleotide sequence coding for RFC40,including as set out in FIG. 18 (SEQ ID NO:2). Particular embodimentsthereof are provided herein, including as set out in FIG. 18 and in FIG.24 and provided in miRNAS of miR-hsa-125a-3p (SEQ ID NO:7), modifiedmiR#1 (SEQ ID NO:8), modified miR#2 (SEQ ID NO:9), modifiedhsa-miR-125a-3p #3 is SEQ ID NO:18, and modified hsa-miR-125a-3p #4 isSEQ ID NO:19, and in siRNAs RFC40-siRNA-S1 (SEQ ID NO:3), RFC40-siRNA-S2(SEQ ID NO:4), RFC40-siRNA-S3 (SEQ ID NO:5) and RFC40-siRNA-S4 (SEQ IDNO:6). The disclosure also comprises shRNA-based targeting of cancercells comprising introducing into cancer cells at least onepolynucleotide which comprises or consists ofACUACGAACUGCCGUGGGUUGAAAAAUAU (SEQ ID NO:15),GUCCCGCUGUGCAGUCCUCCGGUACACAA (SEQ ID NO:16), ACUACGAACUGCCGUGGGUUG (SEQID NO:20), ACUACGAACUGCCGUGGGUUGNNNNNNNNNCAACCCACGGCAGUUCGUAGU (SEQ IDNO:21/hRFC2-shRNA#1 sequence) wherein the NNNNNNNNN may form a loopsequence in an shRNA,GUCCCGCUGUGCAGUCCUCCGGUACACAANNNNNNNNNUGUGUACCGGAGGA CUGCACAGCGGGAC (SEQID NO:24/hRFC2-shRNA#2) wherein the NNNNNNNNN may form a loop sequencein an shRNA. In embodiments the loop sequence can be 7, 9 or 11nucleotides in length. One example of a suitable loop sequence comprisesor consists of UUCAAGAGA (SEQ ID NO: 22). Thus, the disclosure includesuse of shRNA sequences comprising the loop sequence of SEQ ID NO: 22,such as ACUACGAACUGCCGUGGGUUGUUCAAGAGACAACCCACGGCAGUUCGUAGU (SEQ ID NO:23/hRFC2-shRNA#1) andGUCCCGCUGUGCAGUCCUCCGGUACACAAUUCAAGAGAUUGUGUACCGGAGGA CUGCACAGCGGGAC(SEQ ID NO: 25/hRFC2-shRNA#2 sequence.) Such shRNA sequences may alsocomprise a suitable transcription termination signal, such as a poly Tsequence (which can be poly U, such as UUUUUU in shRNA). Additionalsequences may also be part of, or contiguous with, the shRNA sequence,such as expression-vector derived sequences, or other sequences thatwill be apparent to those skilled in the art given the benefit of thepresent disclosure, provided the sequences retain their intendedfunction. As described elsewhere herein, all polynucleotide sequencesand vectors encoding and/or expressing them are included in theinvention. The disclosure includes the inverse, complementary, and DNAand RNA equivalents of every polynucleotide sequence disclosed herein.

It will be recognized by those skilled in the art that polynucleotidesof this disclosure can be introduced into cells and to individuals inneed thereof in a variety of ways. For example, the polynucleotides canbe introduced directly, such as by direct delivery of RNApolynucleotides, whether single stranded, double stranded, or partiallysingle stranded or partially double stranded, or they can be introducedinto an individual and into cancer cells by way of a vector encoding theRNA polynucleotides, such as by using any suitable expression vector,examples of which are known in the art and are publicly available. Inembodiments, a complex of two distinct RNA polynucleotides partially orfully hybridized to one another is used. In various embodiments RNApolynucleotides can be introduced into an individual using an expressionvector that encodes the polynucleotides. In certain cases the expressionvector is a modified virus, and/or a modified viral vector, such as avector that comprises a modified viral genome or segment thereof. Thedisclosure includes introducing into an individual a viral constructencoding one or more polynucleotides of this disclosure. The viralconstructs may comprise a modified retrovirus or modified retroviralgenomic RNA or DNA equivalent thereof, or DNA encoding a particular RNA.In certain non-limiting examples the disclosure includes use of amodified adenovirus or recombinant adeno-associated virus (rAAV). Thedisclosure includes a pharmaceutical composition comprising apolynucleotide encoding one or more of the polynucleotides describedherein. In certain examples the polynucleotide is present in a viralparticle, such as adenovirus particles that have been engineered toexpress, for example, an shRNA of this disclosure.

The down regulation of gene expression using antisense nucleic acids canbe achieved at the translational or transcriptional level. Antisensenucleic acids of the invention are particularly nucleic acid fragmentscapable of specifically hybridizing with all or part of a nucleic acidencoding a target RFC40 polypeptide or the corresponding messenger RNA.In addition, antisense nucleic acids may be designed which decreaseexpression of the nucleic acid sequence capable of encoding a targetRFC40 polypeptide by inhibiting splicing of its primary transcript. Anylength of antisense sequence is suitable for practice of the inventionso long as it is capable of down-regulating or blocking expression of anucleic acid coding for target RFC40. Particularly, the antisensesequence is at least about 15-30, and particularly at least 17nucleotides in length. The preparation and use of antisense nucleicacids, DNA encoding antisense RNAs and the use of oligo and geneticantisense is known in the art. In this regard, the present disclosureprovides a demonstration of using two representative shRNAs to achieve atherapeutic response in clinically pertinent animal models of cancer,and in particular, a mouse model of breast cancer. In this regard, andas shown in FIGS. 27, 28 and 29, and Example 8, administeringhRFC2-shRNA#1, and hRFC2-shRNA#2, is significantly effective in reducingtumor volume (FIG. 29), in female mice challenged with human mammarygland adenocarcinoma xenograft, that represents a triple negative breasttumor, without affecting the body weight (FIG. 28). The shRNAs wereadministered as described in Example 8 using modified adenovirus.

An aspect of these methods relates to the down-regulation or blocking ofthe expression of a target RFC40 polypeptide by the induced expressionof a polynucleotide encoding an intracellular binding protein that iscapable of selectively interacting with the target RFC40 polypeptide. Anintracellular binding protein includes an activity-inhibitory agent andany protein capable of selectively interacting, or binding, with thepolypeptide in the cell in which it is expressed and neutralizing thefunction of the polypeptide. Particularly, the intracellular bindingprotein may be an antibody, particularly a neutralizing antibody, or afragment of an antibody or neutralizing antibody having binding affinityto an epitope of the RFC40 polypeptide, including as set out in FIG. 1(SEQ ID NO:1). In embodiments, the intracellular binding protein is asingle chain antibody.

Various embodiments of these methods comprises the expression-inhibitoryagent selected from the group consisting of antisense RNA, antisenseoligodeoxynucleotide (ODN), a ribozyme that cleaves thepolyribonucleotide coding for RFC40, and a small interfering RNA (siRNA)that is sufficiently homologous to a portion of the RFC40polyribonucleotide as set out in FIG. 18 (SEQ ID NO:2), such that thesiRNA interferes with the translation of the target RFC40polyribonucleotide to the target RFC40 polypeptide.

The present invention also relates to a variety of diagnosticapplications, including methods for detecting the presence, amount oractivity of RFC40 protein, messenger RNA or RFC40 gene amplification, byreference to the RFC40 proteins role in cancer and cancer cellproliferation and division, including in breast cancer. RFC40 antibodiesare known and available in the art, including those utilized in theexemplary methods and assays provided herein and in the examples.Further RFC40 can be used to produce antibodies to itself by a varietyof known techniques, and such antibodies could then be isolated andutilized as in tests for the presence of RFC40 in sample orcancer-suspecting cells.

The RFC40 in cells can be ascertained by the usual immunologicalprocedures applicable to such determinations. A number of usefulprocedures are known, including as employed and utilized in the examplesand studies described herein. Procedures which are useful may utilizeeither the RFC40 labeled with a detectable label, antibody Ab₁ labeledwith a detectable label, or antibody Ab₂ labeled with a detectablelabel. The procedures may be summarized by the following equationswherein the asterisk indicates that the particle is labeled, and “˜”stands for RFC40:˜*+Ab ₁ =˜*Ab ₁  A.˜+Ab*=˜Ab ₁*  B.˜+Ab ₁ +Ab ₂ *=˜Ab ₁ Ab ₂*  C.

The procedures and their application are all familiar to those skilledin the art and accordingly may be utilized within the scope of thepresent invention. The “competitive” procedure, Procedure A, isdescribed in U.S. Pat. Nos. 3,654,090 and 3,850,752. Procedure C, the“sandwich” procedure, is described in U.S. Pat. Nos. RE 31,006 and4,016,043. Still other procedures are known such as the “doubleantibody,” or “DASP” procedure. In each instance, the RFC40 formscomplexes with one or more antibody(ies) or binding partners and onemember of the complex is labeled with a detectable label. The fact thata complex has formed and, if desired, the amount thereof, can bedetermined by known methods applicable to the detection of labels. Thelabels most commonly employed for these studies are radioactiveelements, enzymes, chemicals which fluoresce when exposed to ultravioletlight, and others.

A number of fluorescent materials are known and can be utilized asdetectable labels. These include, for example, fluorescein, rhodamine,auramine, Texas Red, AMCA blue and Lucifer Yellow. A particulardetecting material is anti-rabbit antibody prepared in goats andconjugated with fluorescein through an isothiocyanate. The RFC40 or itsbinding partner(s) can also be labeled with a radioactive element orwith an enzyme. The radioactive label can be detected by any of thecurrently available counting procedures. The preferred isotope may beselected from ³H, ¹⁴C, ³²P, ³⁵S, ³⁶Cl, ⁵¹Cr, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁹⁰Y,¹²⁵I, ¹³¹I, and ¹⁸⁶Re. Enzyme labels are likewise useful, and can bedetected by any of the presently utilized colorimetric,spectrophotometric, fluorospectrophotometric, amperometric or gasometrictechniques. The enzyme is conjugated to the selected particle byreaction with bridging molecules such as carbodiimides, diisocyanates,glutaraldehyde and the like. Many enzymes which can be used in theseprocedures are known and can be utilized. In embodiments the enzymes canbe are peroxidase, β-glucuronidase, β-D-glucosidase, β-D-galactosidase,urease, glucose oxidase plus peroxidase and alkaline phosphatase. U.S.Pat. Nos. 3,654,090; 3,850,752; and 4,016,043 are referred to by way ofexample for their disclosure of alternate labeling material and methods.

In a further embodiment of this invention, commercial test kits suitablefor use by a medical specialist may be prepared to determine thepresence or amount of RFC40 activity, expression or RFC40 geneamplification in suspected cancer cells or biopsy or tumor samples. Oneclass of such kits will contain at least the labeled RFC40 or itsbinding partner, for instance an antibody specific thereto, anddirections, of course, depending upon the method selected, e.g.,“competitive,” “sandwich,” “DASP” and the like. The kits may alsocontain peripheral reagents such as buffers, stabilizers, etc. Inembodiments the kits comprise one or more PCR primers described herein.

Accordingly, a test kit may be prepared for the determination andquantitation of RFC40 protein in cells or a cellular or biopsy sample,comprising:

-   -   (a) a predetermined amount of at least one labeled        immunochemically reactive component obtained by the direct or        indirect attachment of the present RFC40 or a specific binding        partner thereto, to a detectable label;    -   (b) other reagents; and    -   (c) directions for use of said kit.

More specifically, the diagnostic test kit may comprise:

-   -   (a) a known amount of the RFC40 as described above (or a binding        partner) generally bound to a solid phase to form an        immunosorbent, or in the alternative, bound to a suitable tag,        or plural such end products, etc. (or their binding partners)        one of each;    -   (b) if necessary, other reagents; and    -   (c) directions for use of said test kit.        In a further variation, the test kit may be prepared and used        for the purposes stated above, and comprises:    -   (a) a labeled component which has been obtained by coupling the        RFC40 to a detectable label;    -   (b) one or more additional immunochemical reagents of which at        least one reagent is a ligand or an immobilized ligand, which        ligand is selected from the group consisting of:        -   (i) a ligand capable of binding with the labeled component            (a);        -   (ii) a ligand capable of binding with a binding partner of            the labeled component (a);        -   (iii) a ligand capable of binding with at least one of the            component(s) to be determined; and        -   (iv) a ligand capable of binding with at least one of the            binding partners of at least one of the component(s) to be            determined; and    -   (c) directions for the performance of a protocol for the        detection and/or determination of one or more components of an        immunochemical reaction between the RFC40 and a specific binding        partner thereto.

In accordance with the above, an assay system for screening potentialdrugs effective to modulate the activity or expression of RFC40 may beprepared and is provided. The RFC40 may be introduced into a testsystem, and the prospective drug may also be introduced into theresulting cell culture, and the culture thereafter examined to observeany changes in the RFC40 activity of the cells, or in the proliferationor division of the cells, due either to the addition of the prospectivedrug alone, or due to the effect of added quantities of the known RFC40.

The invention may be better understood by reference to the followingnon-limiting Examples, which are provided as exemplary of the inventionwhich are presented in order to more fully illustrate embodiments of thedisclosure and should in no way be construed as limiting the scope ofthe invention.

EXAMPLE 1 RFC40 is a Molecular Marker for Breast Cancer

Cancers with gene copy number amplification or chromosome polysomyusually are the most aggressive types, often associated and correlatedwith invasion, metastasis and recurrence, and cannot be corrected at thechromosomal level (Mizutani T et al (1993) Cancer 72(7):2083-2088;Birkeland E et al (2012) Br J Cancer 107(12):1997-2004; Vlajnic T et al(2011) Modern Pathology 24:1404-1412; Lundgren K et al (20120 BreastCancer Res 14(2):R57). In such cases a strategy for effective cancertreatment is early detection and development of potent chemotherapeuticdrugs that will selectively target protein(s) in the cancerous cells.

In this disclosure, we validate RFC40 as a non-receptor based molecularmarker for breast cancers specifically, estrogen positive (ER positive),estrogen negative (ER negative), and progesterone, estrogen and humanepidermal growth factor receptor 2-HER2 negative or triple negativebreast cancers (TNBC). The assembly of the RFC complex is the first stepin DNA elongation, committing the cell for DNA replication and henceover-expression of RFC40 could be an early event in the development ofcancer and can prove to be a novel early diagnostic marker for theprogression of cancer. Moreover, RFC40 may function as a non-invasivediagnostic tool since it can be detected in blood in blood cancers suchas acute and chronic myeloid leukemia (Staber P B et al (2004) Oncogene23(4):894-904; Merkerova M et al (2007) Neoplasma 54(6):503-10).

Over-expression of any protein(s) in cancer can occur either due to (a)up-regulation of its mRNA, (b) increase in mRNA stability, (c) decreasein the protein degradation rate, and (d) increase in the translationrate. We propose that RFC40 protein and message is over-expressed inbreast cancers, as illustrated in the schematic (FIG. 1). The presentstudies demonstrate that RFC40 protein is over-expressed in breastprimary breast cancer cell lines as well as in patient breast cancertissues.

We determined whether RFC40 is over-expressed in breast cancer usingWestern blot analysis for protein levels and quantitative RT-PCR of mRNAlevels.

Western Blot Studies: We performed western blot analyses for RFC40protein with lysates obtained from non-cancerous mammary epithelialcells (MCF10A), ER positive breast cancer cells (MCF7), ER negativebreast cancer cells (MDA-MB-231) and triple negative breast cancer-likecells (MDA-MB-468) using goat anti-RFC40 antibody (Bethyl Laboratories,TX, USA; Cat#A300-142A) as described previously (Ata H et al (2012) PLoSOne 7(6):e39009). RFC40 was significantly up-regulated in all the breastcancer cells lines as compared to the non-cancerous breast cells (FIG.2), suggesting that RFC40 is over-expressed in these breast cancercells. β-Actin (Santa Cruz, Calif., USA; Cat# sc-47778) was used as theloading control.

To determine the expression of RFC40 protein in patient breast tissues,we used a 96-cores patient breast tissue microarray (BTMAs; Pantomics,Inc., CA, USA) containing 36 cases of breast cancers (20-ER positive and15-ER negative) and 12 cases of normal, reactive and benign tumortissues of the breast, in duplicates (FIG. 3). These BTMAs have beenextensively used and well characterized in several studies (Moreira J Met al (2010) Mol Oncol 4(6):539-61; Unger K et al (2010) Endocr RelatCancer 17(1):87-98). The tissues in the microarrays are fixed in 10%neutral buffered formalin for 24 to 48 hours. Tissue sections were cutfresh upon receiving an order. Pantomics provided the followinginformation about each of the patient breast tumor samples on the BMTAs:(i) sex; (ii) age; (iii) pathology/location; (iv) grade; and (v)staining score for androgen (AR), estrogen (ER), progesterone (PR)receptors and human epidermal growth factor receptor (HER2) byimmunohistochemical analyses (TABLE 1).

TABLE 1 Pathology NO Sex Age Organ diagnosis Grade TNM AR ER PR HER2Type A1, F 50 Breast Normal 1%, + 2%, ++  2%, +++ — NAT B1 A2, F 47Breast Normal/hyper- 5%, + 1%, +    2%, +++ — NAT B2 plasia A3, F 40Breast Normal/hyper- — — 2%, ++ — NAT B3 plasia A4, F 32 BreastNormal/hyper-  5%, ++ 1%, ++ — — NAT B4 plasia A5, F 50 Breast Granuloma— — — — NAT B5 A6, F 30 Breast Granuloma — — — — NAT B6 A7, F 50 BreastFibrocystic  5%, ++ 1%, ++ — + NAT B7 changes A8, F 43 BreastFibrocystic 2%, + 5%, +   — — NAT B8 changes A9, F 25 BreastFibroadenoma    30%, ++~+++ 30%, +++ 15%, +++ +~+++ Benign B9 A10, F 20Breast Fibroadenoma 20%, ++    30%, ++~+++ 50%, +++ + Benign B10 A11, F23 Breast Fibroadenoma 50%, ++ 80%, +++ 60%, +++ + Benign B11 A12, F 50Breast Fibroadenoma 10%, +  15%, ++  30%, +++ + Benign B12 C1, F 43Breast Invasive I TisN0M0 — — — +++ Malig- D1 ductal nant carcinoma C2,F 60 Breast Invasive I TisN0M0 — — — +++ Malig- D2 ductal nant carcinomaC3, F 37 Breast Invasive I TisN0M0 10%, ++    80%, ++~+++    50%, ++~++++~++ Malig- D3 ductal nant carcinoma C4, F 41 Breast Lobular I TisN0M0 —20%, ++  80%, +++ + Malig- D4 carcinoma in nant situ C5, F 30 BreastPhyllodes — — — — Malig- D5 sarcoma nant C6, F 48 Breast Invasive IIT1N0M0 50%, ++    60%, ++~+++ 80%, +++ — Malig- D6 ductal nant carcinomaC7, F 44 Breast Invasive II~III T1N0M0 — — — +++ Malig- D7 ductal nantcarcinoma C8, F 61 Breast Invasive II~III T1N0M0   30%, +~++ 100%, +++ 50%, ++  + Malig- D8 ductal nant carcinoma C9, F 40 Breast InvasiveII~III T1N0M0   30%, +~++ 60%, ++     80%, ++~+++ ++ Malig- D9 ductalnant carcinoma C10, F 38 Breast Invasive I~II T1N0M0 15%, +  15%, +  30%, ++  ++~+++ Malig- D10 ductal nant carcinoma C11, F 43 BreastInvasive III T2N0M0 — 20%, +   30%, ++  — Malig- D11 ductal nantcarcinoma C12, F 48 Breast Invasive II T2N0M0 —  50%, +~++    80%,++~+++ — Malig- D12 ductal nant carcinoma E1, F 62 Breast InvasiveII~III T2N0M0 — 90%, +++ 60%, +++ + Malig- F1 ductal nant carcinoma E2,F 47 Breast Invasive II~III T2N1M0 — 50%, ++  30%, ++  + Malig- F2ductal nant carcinoma E3, F 59 Breast Invasive III T2N0M0 — — — — Malig-F3 ductal nant carcinoma E4, F 72 Breast Invasive I~II T2N1M0 — — — —Malig- F4 ductal nant carcinoma E5, F 36 Breast Invasive III T2N0M0 — —— +++ Malig- F5 ductal nant carcinoma E6, F 48 Breast Invasive II~IIIT2N0M0 — — — +++ Malig- F6 ductal nant carcinoma E7, F 44 BreastInvasive II~III T2N0M0 —  50%, +~++ — — Malig- F7 ductal nant carcinomaE8, F 56 Breast Invasive I~II T2N0M0  5%, ++ — — +++ Malig- F8 ductalnant carcinoma E9, F 50 Breast Invasive II T2N0M0 — — — ++~+++ Malig- F9ductal nant carcinoma E10, F 50 Breast Invasive II~III T2N0M0  5%, ++80%, +++ 80%, +++ + Malig- F10 ductal nant carcinoma E11, F 83 BreastInvasive II T2N0M0 10%, ++ 80%, +++ 50%, ++  + Malig- F11 ductal nantcarcinoma E12, F 64 Breast Invasive III T2N0M0 —    80%, ++~+++ 20%, ++ — Malig- F12 ductal nant carcinoma G1, F 58 Breast Invasive II T2N1M0 —— — Malig- H1 ductal nant carcinoma G2, F 32 Breast Invasive II~IIIT3N0M0 — — 20%, ++  ++~+++ Malig- H2 ductal nant carcinoma G3, F 60Breast Invasive II~III T3N0M0 — — — +++ Malig- H3 ductal nant carcinomaG4, F 58 Breast Invasive III T3N0M0 — — — — Malig- H4 ductal nantcarcinoma G5, F 54 Breast Invasive II~III T3N3M0 — — — ++~+++ Malig- H5ductal nant carcinoma G6, F 33 Breast Invasive III T3N0M0 20%, ++ 80%,+++ 50%, ++  — Malig- H6 ductal nant carcinoma G7, F 51 Breast InvasiveII~III T4N2MX 30%, ++ 80%, +++ — — Malig- H7 ductal nant carcinoma G8, F55 Breast Invasive III T4N2MX — 80%, +++ 50%, +++ — Malig- H8 ductalnant carcinoma G9, F 36 Breast Invasive II~III T4N3MX 30%, ++ 60%, ++    60%, ++~+++ +++ Malig- H9 ductal nant carcinoma G10, F 42 BreastInvasive II~III T4N3MX 2%, +  50%, +~++ — +~++ Malig- H10 ductal nantcarcinoma G11, F 36 Breast Invasive II T4N2M0  5%, ++ 90%, +++ 100%,+++  + Malig- H11 ductal nant carcinoma G12, F 36 Breast Invasive I~IIT4N1M0 — — — +++ Malig- H12 ductal nant carcinoma

The 96 cores patient BTMAs was subjected to immunohistochemical analyses(method in accordance with Golden T et al (2008) Biochim Biophys Acta1782(4):259-70) using polyclonal anti-RFC40 (Bethyl Laboratories, TX,USA; Cat#A300-142A) followed by incubation with HRP-conjugated secondaryantibodies for 1 h. Images of the stained sections were collected usingDako Cytomation system (FIG. 4A). The graph represents the RFC40staining scores (intensity of staining×percentage of cells stained) innormal, estrogen positive (ER⁺) and estrogen negative (ER⁻) samples(FIG. 4B). The RFC40 staining scores were significantly increased inboth positive and negative ER samples versus normal. We found that RFC40protein was up-regulated by 7.9-fold in ER positive and 10.9-fold in ERnegative breast tumors as compared to normal breast tissues (FIG. 4B),indicating that the RFC40 protein was over-expressed in patient breastcancers. Using this data we were able to correlate the grade andpathology of the breast cancers to the over-expression of RFC40 proteinwhich may indicates that RFC40 is a prognostic indicator for therapy(TABLE 2).

TABLE 2 NO Pathology diagnosis TNM IHC for RFC40 Type A1, B1 Normal   2,1.8 Normal A2, B2 Normal/hyperplasia 0.02, 0.03 Normal A4, B4Normal/hyperplasia 0.03, 0.08 Normal A7, B7 Fibrocystic changes 0, 0Normal A8, B8 Fibrocystic changes 0, 0 Normal A9, B9 Fibroadenoma 0.07,0.11 Normal A10, B10 Fibroadenoma 0.13, 0.08 Normal A11, B11Fibroadenoma 0, 0 Normal A12, B12 Fibroadenoma    0, 0.006 Normal C3, D3Invasive ductal carcinoma TisN0M0 13.2, 0.13 ER +ve/HER2+ve C4, D4Lobular carcinoma in situ TisN0M0 4.2, no score ER +ve/HER2+ve C6, D6Invasive ductal carcinoma T1N0M0 0.04, 0.15 ER +ve/HER2−ve C8, D8Invasive ductal carcinoma T1N0M0  0.06, 0.034 ER +ve/HER2+ve C9, D9Invasive ductal carcinoma T1N0M0 0.005, 0.005 ER +ve/HER2+ve C10, D10Invasive ductal carcinoma T1N0M0   9, 2.9 ER +ve/HER2+ve C12, D12Invasive ductal carcinoma T2N0M0 0.01, 0.94 ER +ve/HER2−ve E1, F1Invasive ductal carcinoma T2N0M0 2.4, 5.6 ER +ve/HER2+ve E2, F2 Invasiveductal carcinoma T2N1M0 0.13, 1   ER +ve/HER2+ve E7, F7 Invasive ductalcarcinoma T2N0M0  0.2, 0.05 ER +ve/HER2−ve E10, F10 Invasive ductalcarcinoma T2N0M0 0.29, 1.2  ER +ve/HER2+ve E11, F11 Invasive ductalcarcinoma T2N0M0 0.27, 0.04 ER +ve/HER2+ve E12, F12 Invasive ductalcarcinoma T2N0M0 4.1, 1.7 ER +ve/HER2−ve G6, H6 Invasive ductalcarcinoma T3N0M0 0, 0 ER +ve/HER2−ve G7, H7 Invasive ductal carcinomaT4N2MX  1.6, 1.14 ER +ve/HER2−ve G8, H8 Invasive ductal carcinoma T4N2MX  0, 6.7 ER +ve/HER2−ve G9, H9 Invasive ductal carcinoma T4N3MX 0.042,13.3  ER +ve/HER2+ve G10, H10 Invasive ductal carcinoma T4N3MX 0.47, 0  ER +ve/HER2+ve G11, H11 Invasive ductal carcinoma T4N2M0 0, 0 ER+ve/HER2+ve C1, D1 Invasive ductal carcinoma TisN0M0 0.015, 0    ER−ve/HER2+ve C2, D2 Invasive ductal carcinoma TisN0M0 2.4, 4.5 ER−ve/HER2+ve C7, D7 Invasive ductal carcinoma T1N0M0 0.42, 0.43 ER−ve/HER2+ve E3, F3 Invasive ductal carcinoma T2N0M0 0.009, 0.01  ER−ve/HER2−ve E4, F4 Invasive ductal carcinoma T2N1M0  8.7, 0.01 ER−ve/HER2−ve E5, F5 Invasive ductal carcinoma T2N0M0  0.2, 0.06 ER−ve/HER2+ve E6, F6 Invasive ductal carcinoma T2N0M0 0.07, 0.03 ER−ve/HER2+ve E8, F8 Invasive ductal carcinoma T2N0M0  0.11, 0.005 ER−ve/HER2+ve E9, F9 Invasive ductal carcinoma T2N0M0 11.1, 9.6  ER−ve/HER2+ve G1, H1 Invasive ductal carcinoma T2N1M0 0, 0 ER −ve/HER2−veG2, H2 Invasive ductal carcinoma T3N0M0 0.85, 22.2 ER −ve/HER2+ve G3, H3Invasive ductal carcinoma T3N0M0 12.6, 3.8  ER −ve/HER2+ve G4, H4Invasive ductal carcinoma T3N0M0 1.7, 0   ER −ve/HER2−ve G5, H5 Invasiveductal carcinoma T3N3M0 0.15, 0.06 ER −ve/HER2+ve G12, H12 Invasiveductal carcinoma T4N1M0 0.01, 0   ER −ve/HER2+ve

RFC40 was specifically over-expressed versus other RFC complex proteins.MCF10A, MCF7 and MDA-MB-231 cells were lysed and 35 μg of total proteinlysates were analyzed on 10% SDS-polyacrylamide gels. Protein expressionof RFC40, RFC37 (antibody from Santa Cruz Biotech, CA, USA;Cat#sc-28301) and RFC36 (antibody from Santa Cruz Biotech, CA, USA;Cat#sc-20997) was examined by Western blot analyses, with β-Actin usedas a loading control (FIG. 5). Only RFC40 was increased in the breastcancer cell lines. The other RFC protein levels were comparable in allthree lines tested, as was β-actin level.

mRNA Studies: We performed quantitative RT-PCR for RFC40-mRNA usingtotal RNA (t-RNA) extracted from MCF10A, MCF7, MDA-MB-231 and MDA-MB-468cells as described previously (Ata H et al (2012) PLoS One 7(6):e39009).Assays for quantification of RFC40 and GAPDH mRNA expression wereconducted on the iCycler (BioRad) using specific primers (all primerswere purchased from Invitrogen) as follows: (a) for RFC40—ForwardPrimer: 5′ ATGGAGGTGGAGGCCGTCTGTG3′ (SEQ ID NO:10; Tm=61.9° C.) andReverse Primer: 5′ CCTCTAGCCTGCTCACGGTGTCTTC3′ (SEQ ID NO:11; Tm=61.4°C.); (b) for GAPDH—Forward Primer: 5′ CTCATGACCACAGTCCATGCCATC3′ (SEQ IDNO:12) and Reverse Primer: 5′ CGGAAGGCCATGCCAGTGAG3′ (SEQ ID NO:13).RFC40, and GAPDH mRNA/cDNA amplification was programmed at 55° C. for 10min for cDNA synthesis followed by 95° C. for 5 min (RT enzymeinactivation), and 40 cycles of 95° C. for 10 s, 60° C. for 30 s, and72° C. for 30 s (data collection point). Melting curve analysis wassubsequently conducted in order to verify the purity of the products.The fold increase in the mRNA levels were calculated from the crossingpoint (Ct) deviation of all the samples and normalized with GAPDHvalues. Amplified products were visualized on 4% agarose gels (FIG. 6A).Using ER positive and ER negative breast cancer cells, we found that themessage for RFC40 was up-regulated by 3.86-fold in ER positive (MCF7)and 5.97-fold in ER negative (MDA-MB-231) breast cancer cell lines ascompared to normal-cancerous (MCF10A) breast cells (FIG. 6B),respectively, suggesting that the RFC40 protein over-expression in theER positive and negative breast cancer cells was due to up-regulation inthe RFC40 message. Similarly, the message for RFC40 was significantlyup-regulated by 2 fold in the TBNC-like breast cancer (MDA-MB-468) cellsas compared to non-cancerous breast cells (FIG. 6C), suggesting that theRFC40 protein over-expression in the TNBC-like cells was due toup-regulation in RFC40 message.

EXAMPLE 2 RFC40 is Localized in the Nucleus

It was determined that the pre-dominant localization of RFC40 in thenucleus is an indicator of the highly proliferative state of cancercells. MCF10A, MCF7 and MDA-MB-231 cells were fixed and subjected toimmunofluorescence microscopy using polyclonal anti-RFC40 antibodyfollowed by incubation with Alexa-488-conjugated secondary antibodiesfor 1 has described previously (Gupte R et al (2005) Cancer Biology andTherapy 4(4):429-437). Images of the stained sections were collectedusing a Nikon A1 microscope with Plan ×40/NA 0.25 Phi objective. Nuclearstaining of RFC40 was more pronounced and significant in the breastcancer cells versus normal-cancerous breast cells (FIG. 7A). MCF10A,MCF7 and MDA-MB-231 cells were then subjected to flow cytometricanalyses using DAPI (MCF10A) and PI (MCF7 and MDA-MB-231), respectively.Histograms representing the percent of cells in G1, S and G2 phasesrespectively demonstrate significantly higher percentage of S and G2phase cells in the breast cancer cell lines as compared to non-cancerousbreast cells (FIG. 7B), indicating that nuclear localization of RFC40directly co-relates with increased proliferation in the breast cancercells.

Immunohistochemical analyses of the 96-cores patient BTMA (as performedin Example 1 above) also demonstrated more intense nuclear staining ofRFC40 protein in the ER positive and ER negative as compared to thenormal breast tissue (FIG. 8), suggesting that increased nuclearlocalization of RFC40 may function as an indicator of progression andmetastatic status of breast cancer.

EXAMPLE 3 The RFC40 Gene is Amplified in Breast Cancer

Over-expression of any protein(s) in cancer can occur due to aberrantamplification in its gene copy numbers in addition to up-regulation ofits message and hence protein. Interestingly, amplification of RFC40gene copy numbers as been previous demonstrated in glioblastomas(Nakahara Y et al (2004) Neuro Oncol 6(4):281-9; Suzuki T et al (2004)Brain Tumor Pathol 21(1):27-34). However, whether RFC40 gene copynumbers are increased in breast cancers has not been investigated. Wepropose that RFC40 gene copy number is amplified in breast cancers, asillustrated in the schematic (FIG. 9).

We performed qualitative gene copy number analyses for the RFC40promoter region located on the chromosome 7 at q11.23 position. Weisolated genomic DNA from Adult normal breast tissue, MCF10A andMDA-MB-468 cells and performed qualitative PCR to amplify a 206 bpfragment on the RFC40 promoter and analyzed it on 4% agarose gel. Thedata suggested that the 206 bp fragment on the RFC40 promoter wassignificantly amplified in the MDA-MB-468 cells as compared to normalbreast tissue and non-cancerous breast cells (FIG. 10). This dataindicates that the copy numbers of RFC40 gene is amplified in theTNBC-like cells.

We anticipated that FISH analyses would demonstrate that there isamplification of the RFC40 gene and/or polysomy of chromosome 7 inpatient breast cancer tissues. To determine whether RFC40 gene copynumbers are amplified, Fluorescent in situ hybridization (FISH) inpatient BTMAs (same methods as used in Example 1) was performed using aprobe that hybridized to the RFC40 promoter region (RED; EmpireGenomics, NY, USA) located on chromosome 7 at q11.23 position and aninternal control-chromosome enumeration probe 7 (CEP7; GREEN; Vysisprobes, Abbot, Ill., USA) as described previously (Ata H et al (2012)PLoS One 7(6):e39009). FISH stained slides were visualized bypathologists and the data was analyzed and scored following EGFR-basedmethods as described previously (Varella-Garcia M et al (2009) J ClinPathol 62(11): 970-7). We first observed RFC40 gene copy numberamplification (FIG. 11A) as well as polysomy for chromosome 7 (FIG. 11B)in several estrogen positive and negative breast tumor samples (TABLE3), suggesting that the RFC40 gene is amplified in patient breastcancers.

TABLE 3 FISH SCORES FISH SCORES NO Pathology diagnosis TNM RFC40 CEP7RFC40 CEP7 Type A1, B1 Normal A01-2 2 B01-2 2 Normal A2, B2Normal/hyperplasia A02-2 2 B02-2 2 Normal A4, B4 Normal/hyperplasiaA04-2 2 B04-2 2 Normal A5, B5 Granuloma A05-2 2 B05-2 2 Normal A6, B6Granuloma A06-2 2 B06-2 2 Normal A7, B7 Fibrocystic changes A07-2 2B07-2 2 Normal A8, B8 Fibrocystic changes A08-2 2 B08-2 2 Normal A10,B10 Fibroadenoma A10-2 2 B10-2 2 Normal A11, B11 Fibroadenoma A11-2 2B11-2 2 Normal A12, B12 Fibroadenoma A12-2 2 B12-2 2 Normal C3, D3Invasive ductal carcinoma TisN0M0 C03-2 2 D03-NA NA ER + ve/HER2 + veC4, D4 Lobular carcinoma in situ TisN0M0 C04-2 2 D04-3 3 ER + ve/HER2 +ve C6, D6 Invasive ductal carcinoma T1N0M0 C06-5 3 D06-4 4 ER + ve/HER2− ve C8, D8 Invasive ductal carcinoma T1N0M0 C08-2 2 D08-2 2 ER +ve/HER2 + ve C9, D9 Invasive ductal carcinoma T1N0M0 C09-2 2 D09-2 2ER + ve/HER2 + ve C10, D10 Invasive ductal carcinoma T1N0M0 C10-NA NAD10-3 3 ER + ve/HER2 + ve C12, D12 Invasive ductal carcinoma T2N0M0C12-6 6 D12-3 3 ER + ve/HER2 − ve E1, F1 Invasive ductal carcinomaT2N0M0 E01-2 2 F01-NA NA ER + ve/HER2 + ve E2, F2 Invasive ductalcarcinoma T2N1M0 E02-3 3 F02-2 2 ER + ve/HER2 + ve E7, F7 Invasiveductal carcinoma T2N0M0 E07-2 2 F07-2 2 ER + ve/HER2 − ve E10, F10Invasive ductal carcinoma T2N0M0 E10-2 2 F10-3 3 ER + ve/HER2 + ve E11,F11 Invasive ductal carcinoma T2N0M0 E11-2 2 F11-2 2 ER + ve/HER2 + veE12, F12 Invasive ductal carcinoma T2N0M0 E12-2 2 F12-2 2 ER + ve/HER2 −ve G6, H6 Invasive ductal carcinoma T3N0M0 G06-2 2 H06-2 2 ER + ve/HER2− ve G7, H7 Invasive ductal carcinoma T4N2MX G07-4 4 H07-4 4 ER +ve/HER2 − ve G8, H8 Invasive ductal carcinoma T4N2MX G08-2 2 H08-2 2ER + ve/HER2 − ve G9, H9 Invasive ductal carcinoma T4N3MX G09-3 3 H09-33 ER + ve/HER2 + ve G10, H10 Invasive ductal carcinoma T4N3MX G10-3 3H10-4 4 ER + ve/HER2 + ve G11, H11 Invasive ductal carcinoma T4N2M0G11-2 2 H11-3 3 ER + ve/HER2 + ve C1, D1 Invasive ductal carcinomaTisN0M0 C01-2 2 D01-4 4 ER − ve/HER2 + ve C2, D2 Invasive ductalcarcinoma TisN0M0 C02-2 2 D02-4 4 ER − ve/HER2 + ve C7, D7 Invasiveductal carcinoma T1N0M0 C07-5 5 D07-4 4 ER − ve/HER2 + ve E3, F3Invasive ductal carcinoma T2N0M0 E03-2 2 F03-2 2 ER − ve/HER2 − ve E4,F4 Invasive ductal carcinoma T2N1M0 E04-5 4 F04-2 2 ER − ve/HER2 − veE5, F5 Invasive ductal carcinoma T2N0M0 E05-2 2 F05-2 2 ER − ve/HER2 +ve E6, F6 Invasive ductal carcinoma T2N0M0 E06-5 5 F06-4 4 ER −ve/HER2 + ve E8, F8 Invasive ductal carcinoma T2N0M0 E08-6 2 F08-5 2 ER− ve/HER2 + ve E9, F9 Invasive ductal carcinoma T2N0M0 E09-2 2 F09-2 2ER − ve/HER2 + ve G1, H1 Invasive ductal carcinoma T2N1M0 G01-2 2 H01-33 ER − ve/HER2 − ve G2, H2 Invasive ductal carcinoma T3N0M0 G02-2 2H02-2 2 ER − ve/HER2 + ve G3, H3 Invasive ductal carcinoma T3N0M0 G03-55 H03-3 3 ER − ve/HER2 + ve G4, H4 Invasive ductal carcinoma T3N0M0G04-2 2 H04-2 2 ER − ve/HER2 − ve G5, H5 Invasive ductal carcinomaT3N3M0 G05-4 4 H05-6 6 ER − ve/HER2 + ve G12, H12 Invasive ductalcarcinoma T4N1M0 G12-5 5 H12-4 4 ER − ve/HER2 + ve

EXAMPLE 4 Over-Expression of RFC40 Confers Growth Advantages toNon-Cancerous Breast Epithelial Cells

RFC40 is required for DNA replication, DNA checkpoint repair, genomicstability and sister chromatid cohesion in the cell (Majka J et al(2004) Prog Nucleic Acid Res Mol Biol 78: 227-260; Petronczki M et al(2004) J Cell Sci 117(Pt 16): 3547-3559). Additionally, we have recentlydiscovered that RFC40 is required for accurate chromosomal segregationand completion of cell division after mitosis in proliferating neonatalrat cardiac myocytes, suggesting a role for RFC40 in mitosis andcytokinesis (Ata H et al (2012) PLoS One 7(6):e39009). We also observedthat inhibition of endogenous RFC40 in proliferating neonatal ratcardiac myocytes causes cell death. Consistently, it has beendemonstrated that deletion of RFC40 gene is embryonically lethal inyeast (Cullmann G et al (1995) Mol Cell Biol 15(9):4661-71). Takentogether these findings suggest that RFC40 is required for cellproliferation. Since unrestricted proliferation, as observed in thecancerous cells, requires a continuous supply of the DNA replicationproteins, it is possible that over-expression of RFC40 protein may beassociated with deregulation of growth control, leading to malignanttransformation. Consistently, we have observed that RFC40 protein andmessage is up-regulated and its gene copy numbers amplified in ERpositive and negative breast cancers (see Examples above).Over-expression of RFC40 may be responsible for inducing proliferativeadvantages and causing oncogenic transformation to non-cancerous cells.

We sought to determine whether over-expression of RFC40 can induceoncogenic transformation of non-cancerous breast epithelial breast cellsby investigating the phenotypic transitions that are hallmarks ofoncogenic transformations such as epithelial to mesenchymal transitionas illustrated in the schematic (FIG. 12). Specifically we willdetermine the down-regulation of epithelial marker proteins andover-expression of mesenchymal marker proteins accompanied by epitheloidto stromal phenotypic changes and growth factor-independentproliferation (Overholtzer M et al (2006) Proc Natl Acad Sci USA103(33):12405-10; Kalluri R and Weinberg R A (2009) J Clin Invest119(6):1420-8). In these studies, we over-expressed the RFC40 gene innon-cancerous breast epithelial cells (MCF10A) and determined (I) thepercentage of S-phase cells and cell number analyses to determinewhether over-expression of RFC40 can cause increase in the cell numbers;and (II) changes in cell morphogenesis.

To determine whether over-expression of RFC40 influences the number ofcells in the S-phase, we cloned full-length RFC40 gene in-frame withGreen Fluorescent Protein in adenoviral vector (RFC40-Ad) andtransiently transfected it in MCF10A cells for 48 hr (FIG. 13) asdescribed previously (Gupte R S et al (2011) Antioxid Redox Signal14(4):543-58). Fluorescent assisted cell sorting (FACS) analysis wasperformed to determine the percentage of S-phase cells using univariateanalysis of cellular DNA content in control (GFP-Ad pDNA alone; GFP-Ad)and RFC40 over-expressed MCF10A cells as described previously(Pozarowski P and Darzynkiewicz Z (2004) Methods Mol Biol 281: 301-11).We found that there was increase in the percentage of S-phase cells from3.91% in GFP-Ad (FIG. 14A) to 19.54% in RFC40-Ad over-expressed MCF10Acells (FIG. 14B), with concomitant increase in the percentage of G2/Mphase cells from 10.13% (in GFP-Ad; (FIG. 14A) to 19.77% (in RFC40-Ad;(FIG. 14B) and decrease in the G1-phase cells from 85.96% (in GFP-Ad;(FIG. 14A) to 60.69% (in RFC40-Ad; (FIG. 14B), respectively.Consistently, we observed up-regulation of Cyclin A (S-phase marker) andCyclin B1 (G2/M-phase marker) and down-regulation of Cyclin D1 (G1-phasemarker) in the RFC40-Ad transfected MCF10A cells as compared to GFP-Ad(FIG. 14C), suggesting that over-expression of RFC40 in MCF10A cellspromoted an increase in the number of S-phase cells similar to thoseseen in cancerous cells.

To further assess over-expression of RFC40 and increase in cell number,we transiently transfected MCF10A cells as described above and measuredthe total number of cells using a hemocytometer. We found that there was42.8% increase in the relative cell numbers in RFC40-Ad transfectedMCF10A cells as compared to GFP-Ad (data not shown), suggesting thatover-expression of RFC40 in MCF10A cells promoted proliferation ofRFC40-Ad transfected MCF10A cells.

To determine whether over-expression of RFC40 induces changes in cellmorphogenesis, we transiently transfected MCF10A cells as describedabove, and performed DIC microscopy using a Nikon Eclipse TE2000-E (20×)and found that MCF10A cells transfected with RFC40-Ad appeared to losecell to cell contact and display a stromal-like phenotype (FIG. 15B) ascompared to the epithelial-type phenotype of GFP-Ad transfected MCF10Acells that grew in monolayers (FIG. 15A).

Western blot analysis was then performed to determine whetherover-expression of RFC40 in MCF10A cells promotes the epithelial tomesenchymal transition. Western blot analyses were performed forE-cadherin (down-regulation/loss of epithelial marker) and N-cadherin(gain of mesenchymal markers). We found that E-cadherin wasdown-regulated whereas N-cadherin was up-regulated in RFC40-Adtransfected MCF10A cells as compared to GFP-Ad (FIG. 16), indicatingthat over-expression of RFC40 induced growth advantages tonon-transformed breast epithelial cells and promoted epithelial tomesenchymal transition.

EXAMPLE 5 RFC40 as a Therapeutic Target

Breast cancer accounts for 18% of all cancers in women, making it theforemost cause of cancer-related deaths in women (McPherson K et al(2000) BMJ 321(7261):624-8). Early diagnosis and treatment of breastcancer could play a monumental role in reducing deaths (Misek D E andKim E H (2011) Int J Proteomics 2011:343582). Most of the drugsavailable for the treatment of breast cancers target either theendocrine (estrogen; ER) or growth factor ((ErbB-1, ErbB-2 [humanepidermal growth factor receptor 2; HER2], ErbB-3 and ErbB-4) receptorsfor therapy, however, emerging resistance to endocrine and therapiestargeted against HER2 receptors have created a dire need foridentification of molecular targets that are non-receptor based anddirectly involved in the proliferation of the cancer cells (Normanno Net al (2005) Endocr Relat Cancer 12(4):721-47; Normanno N et al (2009)Endocr Relat Cancer 16(3):675-702). Triple Negative breast cancer(TNBC), a subtype of breast cancer where tumors and cells lack theestrogen, progesterone as well as the human epidermal growth factorreceptor 2 which will not respond to any traditional therapies, isemerging as the most aggressive of breast cancers that can metastasisbeyond the breast and are more likely to recur after treatment.

Prior to the studies in the above Examples, the role of RFC40 in breastcancer had not been assessed. The present studies demonstrate theover-expression of RFC40 protein and RFC40 gene amplification inestrogen positive and negative breast cancers and its role in cellproliferation in cancer. The direct correlation between RFC40over-expression and the progression and metastatic status of breastcancer makes it an effective candidate for a novel non-receptor basedmolecular target for breast cancers. Interestingly, since the DNAreplication machinery does not change irrespective of the tissue type orthe extracellular stimuli, such as endocrine and growth receptors,identifying molecular targets involved in DNA replication such as RFC40may offer a global treatment for all subtypes of breast cancer.Additionally, since there are no reported polymorphisms for the RFC40gene/protein, target-based therapy against this protein will coverbreast cancer treatment across all ethnic groups. Furthermore, itsover-expression in choriocarcinoma (Cui J Q et al (2004) Chinese JCancer 23:196-200) and cancers of various tissues such as acute andchronic myeloid leukemia (Staber P B et al (2004) Oncogene23(4):894-904; Merkerova M et al (2007) Neoplasma 54(6):503-10),nasopharyngeal cancer (Xiong S et al (2011) Med Oncol 28(Suppl1):S341-8), and glioblastomas (which were accidental findings of theirrespective studies) (Nakahara Y et al (2004) Neuro Oncol 6(4):281-9;Suzuki T et al (2004) Brain Tumor Pathol 21(1):27-34), makes RFC40 anovel and universal molecular target for anti-cancer drug therapy.

Furthermore, and without intending to be constrained by any particulartheory, unlike other conventional drugs that globally bind to DNAdirectly, causing inhibition of DNA synthesis as well as DNA repair,targeting RFC40 for drug development would inhibit the formation of theRFC complex, thereby stalling DNA replication without damaging the DNAitself. This approach would provide a novel alternative to conventionaldrugs by significantly minimizing the off-target effects on DNA as wellas other proteins involved in DNA repair.

siRNA and miRNA studies: The data provided herein and below (Example 6and 7) indicate that inhibition of endogenous RFC40 by siRNA or miRNAsis onco-specific and occurs only in the cancerous cells (probably due toup-regulated levels of RFC40-mRNA in the highly proliferative cancercell population) as compared to normal cells. This establishes theunique possibility of inhibition of RFC40 specifically in cancerouscells, thus offering selectivity and specificity for therapeuticintervention. Specifically targeting endogenous RFC40 by blockingtranslation using siRNA, miRNA, as well as via antisense or ribozymeapproaches provides a new and directed approach to breast cancer,whether estrogen sensitive, estrogen resistant or TNBC, and a means toinhibit cell division and growth of cancer cells or tumors, viainhibiting or blocking DNA replication specifically in cancer or tumorcells.

Small molecule compounds: Previous studies have demonstrated that RFC40interacts directly with RIα, which is a regulatory subunit of ProteinKinase A (PKA), and that inhibition of the interaction between RFC40 andRIα, which is required to transport RFC40 into the nucleus, results inG1 arrest (Gupte R et al (2005) Cancer Biology and Therapy4(4):429-437). RIα is a regulatory subunit associated with the PKAI orRI form of PKA, which is a versatile serine-threonine kinase thatmediates cAMP dependent regulation for a variety of cellular processes.Taking into consideration the above examples demonstrating the linkbetween RFC40 and breast cancer and increased expression of RFC40 inbreast cancer, we hypothesized that compounds that will disrupt theRFC40-RIα interaction, thereby preventing transport of RFC40 to thenucleus where it is required for activity, will affect the cell survivalof breast cancer cells.

To assess this, we have treated estrogen positive breast cancer cells(MCF7) with indole-3-carbinol compounds and subjected the cell lysatesto immunoprecipitation experiments using anti-RFC40 antibody, usingmethods as described previously (Gupte R S et al (2005) Cell Cycle 4(2):323-329). We found that the RFC40-RIα interaction was almost completelyabolished in the indole-3-carbinol treated MCF7 cells as compared to thecontrol (data not shown). Indole-3-Carbinol (I3C) is a compound found incruciferous vegetables including broccoli, cabbage and cauliflower.Several studies demonstrate that it can cause cell cycle arrest andapoptosis in cancer cell lines (Wattenburg L W (1978) Cancer Res38:1410-1413; Cover C M et al (1999) Cancer Res 59:1244-1251; Cover C Met al (1998) J Biol Chem 273:3838-3847; Chinni S R (2002) Clin CancerRes 8:1228-1236; Chen D Z et al (2001) J Nutr 131:3294-3302; Hong C etal (2002) Biochem Pharmacol 63(6):1085-1097; Nachshon-Kedmi, M et al(2003) Food Chem Toxicol 41(6):745-752; Choi H S et al (2010)48(3):883-890). Hsu et al. demonstrated that indole-3-carbinol induces aG1 growth arrest of human prostate cancer cells (Hsu J et al (2006)Biochem Pharmacol 72(12):1714-1723).

Other approaches to utilize the RFC40 link in breast cancer fortreatment via inhibiting the RIα interaction with RFC40, or to directlyinhibit RFC40 activity include cAMP modulators and inhibitors ofCDK/cyclin E complex, such as olomoucine. Elevated intracellular cAMPlevels exert transcriptional/post-transcriptional effects on mRNA levelsand a translation effect on the protein expressions of both RFC40 andR1α, thereby increasing the amount of the R1α-RFC40 complex formationand hence promoting the nuclear transport of RFC40 by R1α (Gupte R et al(2006) Exper Cell Res 312:796-806). Once in the nucleus, dissociation ofthe R1α-RFC40 complex requires phosphorylation of R1α by the CDK2/CyclinE complex, as evidenced by the inability of the R1α-RFC40 complex todissociate in the presence of olomoucine(2-(2-hydroxyethylamino)-6-benzylamino-9-methylpurine), a competitiveinhibitor (ATP-binding site) or cyclin dependent kinases, particularlycdc2/cyclinB, cdk2/cyclin A and cdk2/cyclinE. Inability of the R1α-RFC40complexed proteins to dissociate efficiently from each other furtheraffects the ability of RFC40 to form a complex with RFC37 and hence thefunctional RFC pentamer, subsequently affecting DNAsynthesis/replication. Thus olomoucine, or the more efficient inhibitorroscovitine, have applicability in treatment or alleviation of breastcancer, particularly via altering RIα-RFC40 complex.

EXAMPLE 6 siRNA Inhibition of Endogenous RFC40 Results in Cell Death inBreast Cancer Cells

Taking into consideration the above studies and results, it was thenpredicted that inhibition of endogenous RFC40 can inhibit cellsurvival/proliferation and cause either cycle arrest or cell death incancer cells, particularly breast cancer cells. Inhibition of endogenousRFC40 by RFC40-siRNA would target the RFC40 gene in both normal as wellas the cancerous cells, since it is also expressed in normal breastcells. However, univariate analysis of cellular DNA content in MCF10Aand MDA-MB-468 (FIG. 17) cells and MCF7 and MDA-MB-231 (FIG. 7) cellsusing methods described previously (Staber P B et al (2004) Oncogene23(4):894-904), demonstrated that the rate of DNA replication isrelatively low (approximately 3% S-phase cells) in MCF10A cells (FIGS. 7& 17) as compared to MCF7 cells (approximately 33.6% S-phase cells; FIG.7), MDA-MB-231 cells (approximately 16.6% S-phase cells; FIG. 7) andMDA-MB-468 cells (approximately 20-30% S-phase cells; FIG. 17) and alsothat the RFC40 message is maintained at low levels in the MCF10A cellsas compared to MCF7, MDA-MB-231 and MDA-MB-468 cells (FIG. 6). Hence, weanticipated that the effect of RFC40-siRNA on MCF10A cells would be lesspronounced than that in the cancerous cells.

We sought to inhibit the endogenous RFC40 gene by transfecting MCF10A,MCF7, MDA-MB-231 and MDA-MB-468 cells with ON-TargetPLUS Smartpool siRNAagainst RFC40 for 72 hr. The RFC40 mRNA sequence and Smartpool siRNA setof four siRNA sequences are shown in FIG. 15. The Smartpool siRNA(Dharmacon, Inc., TX, USA) is comprised of four RFC40 targeted siRNAsequences (Cat# L-019061-00-0005): RFC40-siRNA-S1 (Cat# J-019061-05),RFC40-siRNA-S2 (Cat# J-019061-06), RFC40-siRNA-S3 (Cat# J-019061-07) andRFC40-siRNA-S4 (Cat# J-019061-08), which targets RFC40-mRNA as shown inFIG. 18. Additionally, cells were also transfected with a scrambledsiRNA sequence/non-targeting-siRNA (NT) (Dharmacon, Inc. Tx, USA; Cat#D-001210-02-05), that does not target for any known human genes as anegative control as shown in FIG. 18.

siRNA transfection Protocol: MCF10A (non-cancerous; FIG. 19A), MCF7(estrogen positive breast cancer cells; FIG. 19B), MDA-MB-231 (estrogennegative breast cancer cells; FIG. 19C) and MDA-MB-468 (TNBC cells; FIG.19D) cells were transfected with non targeting (NT; 100 nM; FIGS. 19A &D), Lamin A/C—(LAC; 100 nM; FIGS. 19B & C),glucose-6-phosphate-dehydrogenase (G6PD; 100 nM; FIG. 19A) and RFC40siRNA-smartpool (100 nM; cocktail of four different sequences; FIG.19A-D) as indicated in the figure for 72 hr using 2.5 μl of DharmafectReagent 1 (Dharmacon, Inc., TX, USA). Cells lysates were subjected toWestern blot analysis using anti-RFC40, anti-RIα (Pharmingen, Inc., CA,USA) and anti-G6PD (Santa Cruz, Calif., USA) antibodies, respectively.β-Actin was used as loading control.

Remarkably, we found that endogenous RFC40 protein was not knocked-downin MCF10A cells (FIG. 19A). To confirm that the MCF10A cells wereaccessible to the siRNA and the transfection reagent, we transfectedMCF10A cells with an on-target siRNA against a house-keeping gene,Glucose-6 phosphate dehydrogenase (G6PD) for 72 hr using identicalexperimental conditions as RFC40-SiRNA. We observed almost 90-95%knock-down of G6PD protein in MCF10A cells (FIG. 19A). Furthermore,RFC40 protein was approximately 85-90% knocked-down in estrogen positivebreast cancer cells (MCF7) and estrogen negative breast cancer cells(MDA-MB-231) (FIGS. 19B and C). Similar specific knock down of RFC40 wasobserved MDA-MB-468 cells (FIG. 19D), suggesting that RFC40 gene/proteinwas selectively not knocked-down only in the cancerous cells.

Furthermore, before lysing the cells we performed cell count analysesusing hemocytometer on all the cell types. The number of each of thecancerous MCF7, MDA-MB-231 and MDA-MB-468 cells was significantlyreduced in siRNA treated versus untreated conditions (FIG. 20), forexample the number of MDA-MB-468 cells was reduced by approximately 60%in the RFC40-siRNA treated cells as compared to those treated with NT(FIG. 20D), suggesting that inhibition of endogenous RFC40 resulted incell death. In striking contrast, there was minimal reduction in thecell numbers in the RFC40-siRNA treated noncancerous MCF10A cells ascompared to those treated with UT (FIG. 20A).

We next incubated untransfected (Unt), NT and RFC40-siRNA treated cellswith Hoechst 33342 that binds to the DNA, for 45 min at 37° C. andperformed immunofluorescent microscopy (at 20×). We found severalapoptotic nuclei in the RFC40-siRNA treated MDA-MB-468 cells (FIG. 21B)as compared to NT or Unt, however, no such apoptotic nuclei was observedin the RFC40-siRNA treated MCF10A cells (FIG. 21A), suggesting that celldeath occurred, and may be due to apoptosis, only in the MDA-MB-468cells, after RFC40-SiRNA treatment. This data indicates that effectivetargeting of RFC40 occurs selectively only in the cancerous cells andnot the normal cells.

To further evaluate the siRNA sequences against RFC40, normal and breastcancer cell lines were transfected with individual siRNAs from theOn-Targetplus-Smartpool selection, particularly with a single siRNA andparticularly either of RFC40-siRNA-S1, RFC40-siRNA-S2, RFC40-siRNA-S3,or RFC40-siRNA-S4, using the transfection protocol as described above.MCF10A and MDA-MB-231 cells were transfected withRFC40-SiRNA-S1/S2/S3/S4 (100 nM) for 72 hr and RFC40 protein expressionwas assessed by Western blot analyses RFC40 protein was significantlyreduced by S1, S2, S3 and S4 siRNA in MDA-MB-231 cells as compared tothat in MCF10A cells (FIG. 22). The β-Actin control was unchanged.

Similar experiments were conducted with individual siRNAs S1, S2, S3 andS4 on MCF10A and MDA-MB-231 cells. Cell number analysis was performedusing Cyquant cell number analyses kit. The results are depicted in FIG.23. The graphs represent the number of MCF10A (FIG. 23A; n=8) andMDA-MB-231 (FIG. 23B; n=8) cells versus the untransfected (UT) andRFC40-siRNA-Si/S2/S3/S4 treated cells. No significant effect to reducecell number was seen in normal MCF10A cells, in fact siRNA S4 appearedto increase cell number somewhat. In contrast, reduction of cell numbersin estrogen negative breast cancer cells MDA-MB-231 cells by each of S2,S3 and S4 was statistically significant.

EXAMPLE 7 miR Studies

To further assess RFC40 inhibition via another approach, studies wereundertaken with miRNA (microRNA, miR). Human miR-hsa-125a-3p aligns withRFC2 (RFC40) sequence (see alignment in FIG. 24) with a mirSVR score of−0.1347 or -0.14 and is listed as a mRNA targeted by hsa-miR-125a-3p,along with numerous other mRNAs in microRNA.org. The microRNA.org sitelists over 9,000 mRNA predicted targets for miR-hsa-125a-3p, with scoresstarting at −2.97 ranging down to −0. No validated targets are listed.MicroRNA target predictions utilize recognized and published methods(Betel, D. et al (2010) Genome Biology 11:R90; Betel, D et al (2008)Nucl Acids Res 36:D149-53; John, B et al (2005) PLoS Biol 3(7):e264).Another microRNA website, the miRDB site, provides 295 predicted targetmRNAs and RFC40 (RFC2) is not among the predicted targets. There is noreport of any specific effect or activity of miR-hsa-125a-3p againstRFC40. Activity would not be predicted as significant, particularlygiven the mirSVR score, and that there are many other targets withhigher relative scores. The miR-hsa-125a-3p sequence is5′ACAGGUGAGGUUCUUGGGAGCC3′ (SEQ ID NO:7) (Dharmacon, Inc. TX, USA; Cat#C-301060-01-0005).

To determine miR-hsa-125a-3p activity against RFC40, human MFC10A andMDA-MB-231 cells were transfected with miRNA negative control#1—(Accession no. MI0000315; Dharmacon, Inc., TX, USA; Cat#CN-002000-01-05) and miR-hsa-125a-3p (100 nM) for 72 hrs. Effects onRFC40 protein levels was determined by Western blot analysis usinganti-RFC40 antibody. β-Actin was used as a loading control. RFC40protein was reduced after transfection of miR-hsa-125a-3p, but notaffected in untreated or miRNA negative control#1 treated MDA-MB-231cells (FIG. 25). No change or difference was observed in MCF10A cells.

Similarly, human MFC10A and MDA-MB-231 cells transfected with miRNAnegative control #1 and miR-hsa-125a-3p (100 nM) for 72 hrs weretrypsinized, resuspended in 1×PBS and counted using a hemocytometer.Cell number was determined for miR treated and untreated cells and isgraphed in FIG. 26. The cell number of MCF10A cells was notsignificantly different in miR treated versus untreated conditions.However, cell number of MDA-MB-231 (estrogen (ER) negative breastcancer) cells significantly decreased on treatment with miR-hsa-125a-3p(FIG. 26).

In order to alter the complementarity of an RFC40 targeted miRNA andfurther test on-target effects, alternative miRs have been designedshowing improved and reduced complementarity for RFC40 (RFC2). Modifiedalignments and sequences are depicted in FIG. 24. An exemplary improvedmiR is provided in ACAGGUGAUCCACUUGGGAGCC (modified miR#1 FIG. 24) (SEQID NO:8).

EXAMPLE 8 In Vivo Studies

This Example provides a demonstration of the efficacy of RNAi treatmentson MDA-MB231 human breast carcinoma xenograft in female NCr nu/nu mice.

The following materials and methods were used to obtain and use theshRNA constructs as indicated in the following description and as setforth in FIGS. 27, 28 and 29.

shRNA construction: shRNAs were constructed by Vector Biolabs, Inc. (PA,USA). Briefly, two short hairpin RNAs (shRNAs) against the human RFC2gene (hRFC2-shRNA#1 and hRFC2-shRNA#2; SEQ ID: X; FIG. 1) weresynthesized, sequenced and PAGE purified. The shRNAs were sub-clonedinto an adenoviral shuttle vector under dual promoters, where thehRFC2-shRNAs were expressed under the control of the human U6 promoterwith a green fluorescent protein (GFP) expression to track thetransfection efficiency under the CMV promoter (Ad-GFP-U6-hRFC2-shRNA#1or 2).

Adenoviral Amplification and purification: Was performed by VectorBiolabs. Briefly, ˜4000 cm² of 293 cell monolayer was infected through aseries of amplification from the above viral and the cells startedshowing cytopathic effects, cells were harvested and lysed to releasethe viruses. The adenoviruses were then be purified by centrifugationusing two sequential cesium chloride gradients. The final product wasdesalted, titrated both spectrophotometrically for viral particles andplaque formation assay for PFU/IFU, and tested for sterility beforeshipment.

Concentration: The final concentration of each of the constructs inparticle forming units (PFU) is as shown in Table 4:

TABLE 4 SR. VP PFU NO CONSTRUCTS TITER-VP/ml TITER-IFU/ml 1 Ad-GFP 4.3 ×10¹² 1.2 × 10¹¹ 2 Ad-GFP-U6-hRFC2-shRNA#1 3.6 × 10¹² 1.0 × 10¹¹ 3Ad-GFP-U6-hRFC2-shRNA#2 5.0 × 10¹² 1.8 × 10¹¹

In Vivo Animal Testing:

in vivo animal testing was performed by Charles River Laboratories (CRL;North Carolina, USA)

Mice

Female athymic nude mice (CRL: NU(NCr)-Foxn1nu, Charles River) were nineweeks old, with a body weight (BW) range of 19.5-26.0 g, on D1 of thestudy. The animals were fed ad libitum water (reverse osmosis, 1 ppm Cl)and NIH 31 Modified and Irradiated Lab Diet® consisting of 18.0% crudeprotein, 5.0% crude fat, and 5.0% crude fiber. The mice were housed onirradiated Enrich-o'cobs™ Laboratory Animal Bedding in staticmicroisolators on a 12-hour light cycle at 20-22° C. (68-72° F.) and40-60% humidity. CR Discovery Services specifically complies with therecommendations of the Guide for Care and Use of Laboratory Animals withrespect to restraint, husbandry, surgical procedures, feed and fluidregulation, and veterinary care. The animal care and use program at CRDiscovery is accredited by the Association for Assessment andAccreditation of Laboratory Animal Care International (AAALAC), whichassures compliance with accepted standards for the care and use oflaboratory animals.

Tumor Cell Culture

The MDA-MB-231 human mammary gland adenocarcinoma cell line wasestablished from a pleural effusion. The cell line is maintained at CRDiscovery Services in RPMI 1640 medium containing 100 units/mLpenicillin G sodium, 100 μg/mL streptomycin sulfate, and 25 μg/mLgentamicin. The medium was supplemented with 10% fetal bovine serum, and2 mM glutamine. The tumor cells were cultured in tissue culture flasksin a humidified incubator at 37° C., in an atmosphere of 5% CO2 and 95%air.

In Vivo Implantation and Tumor Growth

The MDA-MB231 cells used for implantation were harvested during logphase growth and resuspended in cold PBS containing. Each mouse wasinjected subcutaneously in the right flank with 5×106 cells (0.1 mL cellsuspension). Tumors were calipered in two dimensions to monitor growthas their mean volume approached the desired 90 to 130 mm3 range. Tumorsize, in mm3, was calculated from:

${{Tumor}\mspace{14mu}{Volume}} = \frac{w \times l}{2}$where w=width and l=length, in mm, of the tumor. Tumor weight can beestimated with the assumption that 1 mg is equivalent to 1 mm3 of tumorvolume.

Twenty-two days after tumor cell implantation, on Day 1 of the study,animals were sorted into three groups (n=10/group) with individual tumorvolumes of 108 to 144 mm3, and group mean tumor volumes of 113-115 mm3.Tumors were measured with a caliper twice weekly for the duration of thestudy.

Test Articles

AD-GFP (1.2×10¹¹ PFU/mL), Ad-GFP-U6-hRFC2-shRNA#1 (1.0×10¹¹ PFU/ml), andAd-GFP-U6-hRFC2-shRNA#2 (1.8×10¹¹ PFU/mL) viral particles were stored at−80° C. These agents were coded IZ01, IZ02, and IZ03, respectively, forconfidentiality during testing. Each treatment day, a vial was thawedand was used for dosing. Any remaining dose solution was stored at −80°C. and used for the following dose.

Treatment Plan

On D1, mice were sorted into three groups of ten animals and weretreated in accordance with the protocol in Table 2. All agents weredelivered intratumorally (i.tu.), twice weekly for four weeks (biwk×4).Group 1 received 4.56×10⁹ PFU AD-GFP. Groups 2 and 3 received 4.5×10⁹PFUs of Ad-GFP-U6-hRFC2-shRNA#1 or Ad-GFP-U6-hRFC2-shRNA#2,respectively.

Dose volumes were dependent upon the provided dosing solution and wereadministered as follows: AD-GFP (38 uL), Ad-GFP-U6-hRFC2-shRNA (#1) wasdelivered at 45 uL, and Ad-GFP-U6-hRFC2-shRNA#2 was delivered at 25 uL,as shown in Table 5.

TABLE 5 Volume of Group n Agent PFU/animal agent/animal mg/kg RouteSchedule 1 10 IZ01 (AD-GFP) 4.56 × 10⁹  38 μl 4560000000 itu biwk x 4 210 IZ02 4.5 × 10⁹ 45 μl 4560000000 itu biwk x 4 (Ad-GFP-U6-hRFC2-shRNA#1) 3 10 IZ03 (Ad-GFP-U6- 4.5 × 10⁹ 25 μl 4560000000 itu biwk x 4hRFC2-shRNA#2)

Endpoint and Tumor Growth Inhibition (TGI) Analysis

Tumors were measured using calipers twice per week. The study endpointwas defined as a mean tumor volume of 1500 mm3 in the control group or30 days, whichever came first. The study ended on D28. Treatmentefficacy was determined using data from the final day (D28). The MTV(n), the median tumor volume for the number of animals, n, on the finalday, was determined for each group. Percent tumor growth inhibition (%TGI) was defined as the difference between the MTV of the designatedcontrol group (Group 1) and the MTV of the drug treated group, expressedas a percentage of the MTV of the control group:

${\%\mspace{14mu} T\; G\; I} = {{( \frac{{M\; T\; V_{control}} - {M\; T\; V_{{drug} - {treated}}}}{M\; T\; V_{control}} ) \times 100} = {\lbrack {1 - ( {M\; T\; V_{{drug} - {treated}}\text{/}M\; T\; V_{control}} )} \rbrack \times 100}}$

The data set for TGI analysis includes all animals in a group, exceptthose that die due to treatment-related (TR) or non-treatment-related(NTR) causes. CR Discovery Services considers an agent that produces atleast 60% TGI in this assay to be potentially therapeutically active.

Criteria for Regression Responses

Treatment efficacy may also be determined from the incidence andmagnitude of regression responses observed during the study. Treatmentmay cause partial regression (PR) or complete regression (CR) of thetumor in an animal. In a PR response, the tumor volume was 50% or lessof its Day 1 volume for three consecutive measurements during the courseof the study, and equal to or greater than 13.5 mm3 for one or more ofthese three measurements. In a CR response, the tumor volume was lessthan 13.5 mm3 for three consecutive measurements during the course ofthe study.

Toxicity

Animals were weighed daily on Days 1-5, and then on a twice weeklyschedule. The mice were observed frequently for health and overt signsof any adverse treatment related TR side effects, and noteworthyclinical observations were recorded. Individual body weight loss wasmonitored per protocol, and any animal with weight loss exceeding 30%for one measurement, or exceeding 25% for three measurements, was to beeuthanized for health as a TR death. If group mean body weightrecovered, dosing may resume in that group, but at a lower dose or lessfrequent dosing schedule. Acceptable toxicity was defined as a groupmean BW loss of less than 20% during the study and not more than one TRdeath among ten treated animals, or 10%. Any dosing regimen resulting ingreater toxicity is considered above the maximum tolerated dose (MTD). Adeath was to be classified as TR if it was attributable to treatmentside effects as evidenced by clinical signs and/or necropsy, or may alsobe classified as TR if due to unknown causes during the dosing period orwithin 14 days of the last dose. A death was classified as NTR if therewas evidence that the death was related to the tumor model, rather thantreatment related. NTR deaths are further categorized as NTRa (due toaccident or human error), NTRm (due to necropsy-confirmed tumordissemination by invasion or metastasis), and NTRu (due to unknowncauses).

Statistical and Graphical Analyses

Prism (GraphPad) for Windows 6.07 was used for graphical presentationsand statistical analyses.

Two-tailed statistical analyses were conducted at significance levelP=0.05. The Grubb's test was used to check for outliers among the tumorvolumes within groups on D28. Animal 1 of Group 2 and Animal 10 of Group3 were identified as outliers (P<0.05). Prism summarizes test results asnot significant (ns) at P>0.05, significant (symbolized by “*”) at0.01<P≦0.05, very significant (“**”) at 0.001<P≦0.01, and extremelysignificant (“***”) at P≦0.001.

TABLE 6 MTV (n) Statistical Regressions Mean BW Deaths Group n Agent Day28 % TGI Significance PR CR Nadir TR NTR 1 10 IZ01 (AD-GFP)  363 (10) —— 1 0 −0.6% Day 2 0 0 2 10 IZ02 126 (9) 65 *** 2 0 −0.4% Day 2 0 0(Ad-GFP-U6-hRFC2- shRNA#1) 3 10 IZ03 (Ad-GFP-U6- 288 (9) 21 ns 2 0 −1.5%Day 2 0 0 hRFC2-shRNA#2)

Group 2 Animal 1 and Group 3 Animal 10 were excluded from analyses

Study Endpoint=1500 mm³; Study Duration=28 Days

For the Figures and Tables presented in this Example, n=number ofanimals in a group not dead from accidental or unknown causes, oreuthanized for sampling; % TGI=[1−(MTVdrugtreated/MTVcontrol)]×100=percent tumor growth inhibition, compared toGroup 1; Statistical Significance (Two-Way ANOVA): ne=not evaluable,ns=not significant, *=P<0.05, **=P<0.01, ***=P<0.001, compared to Group1, Day 28 TV; MTV (n)=median tumor volume (mm³) for the number ofanimals on the Day of TGI analysis (includes animals with tumor volumeat endpoint); PR=partial regression; CR=complete regression; Mean BWNadir=lowest group mean body weight, as % change from Day 1; ---indicates no decrease in mean body weight was observed;TR=treatment-related death; NTR=non-treatment-related death.

This invention may be embodied in other forms or carried out in otherways without departing from the spirit or essential characteristicsthereof. The present disclosure is therefore to be considered as in allaspects illustrate and not restrictive, the scope of the invention beingindicated by the appended Claims, and all changes which come within themeaning and range of equivalency are intended to be embraced therein.

Various references are cited throughout this Specification, each ofwhich is incorporated herein by reference in its entirety.

What is claimed is:
 1. A kit for diagnosing cancer in an individual,wherein the kit comprises probes for use in a quantitative real timepolymerase chain reaction (qRT-PCR) of RFC40 mRNA, wherein a first probecomprises the sequence of SEQ ID NO:10 and a second probe comprises thesequence SEQ ID NO:11, and wherein the first probe, the second probe, orthe first and second probes are detectably labeled.
 2. The kit of claim1, further comprising at least one sealed container which contains thefirst probe and/or the second probe, the kit further comprising printedmaterial providing instructions on use of the probes for determining anamount of the RFC40 mRNA.
 3. The kit of claim 2, further comprising atleast one additional container, the additional container comprising atleast one buffer for use in the qRT-PCR.
 4. A method of diagnosing andtreating breast cancer in an individual comprising: a) using the kit ofclaim 1, analyzing a sample from the individual comprising breast tissuefor RFC40-mRNA, wherein the patient is diagnosed with breast cancer ifthe RFC40-mRNA is present in an amount greater than a control; and b)administering to the individual a pharmaceutical composition comprisinga polynucleotide, wherein the polynucleotide comprises one or moresequences selected from the group consisting of SEQ ID NO:8; SEQ IDNO:9; SEQ ID NO:18; SEQ ID NO:19; a polynucleotide encoding an shRNAcomprising the sequence of SEQ ID NO:21; a polynucleotide encoding anshRNA comprising the sequence of SEQ ID NO: 24; a polynucleotidecomprising the sequence of SEQ ID NO:21, and a polynucleotide comprisingthe sequence of SEQ ID NO:24.
 5. The method of claim 4, wherein thepolynucleotide encoding the shRNA of SEQ ID NO:21 or the polynucleotideof SEQ ID NO:24 is present in a recombinant virus.
 6. The method ofclaim 4, wherein the NNNNNNNNN segment of SEQ ID NO:21 or SEQ ID NO:24comprises SEQ ID NO:22.
 7. The method of claim 5, wherein the NNNNNNNNNsegment of SEQ ID NO:21 or SEQ ID NO:24 comprises SEQ ID NO:22.